Protein Kinase Modulation by Hops and Acacia Products

ABSTRACT

Botanical compounds to modulate kinase activity are disclosed. The compounds and methods disclosed also inhibit expression of COX-2, inhibit synthesis o prostaglandins selectively in target cells, and inhibit inflammatory response selectively. The compositions contain at least one fraction isolated or derived from hops or  Acacia.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application claims priority to U.S. provisional applicationsSer. No. 60/706,984 filed on Aug. 9, 2005, and Ser. No. 60/748,931 filedon Dec. 9, 2005.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to methods and compositions thatcan be used to treat or inhibit pathological conditions associated withtissue-specific activation of protein kinase activity and/orinflammation, to methods of modulating protein kinase activity in cellsand to methods of modulating inflammation. More specifically, theinvention relates to methods and compositions which utilize extracts,derivatives or fractions isolated either from hops or from members ofthe plant genus Acacia, or combinations thereof.

2. Description of the Related Art

Signal transduction provides an overarching regulatory mechanismimportant to maintaining normal homeostasis or, if perturbed, acting asa causative or contributing mechanism associated with numerous diseasepathologies and conditions. At the cellular level, signal transductionrefers to the movement of a signal or signaling moiety from outside ofthe cell to the cell interior. The signal, upon reaching its receptortarget, may initiate ligand-receptor interactions requisite to manycellular events, some of which may further act as a subsequent signal.Such interactions serve to not only as a series cascade but moreover anintricate interacting network or web of signal events capable ofproviding fine-tuned control of homeostatic processes. This networkhowever can become dysregulated, thereby resulting in an alteration incellular activity and changes in the program of genes expressed withinthe responding cell. See, for example, FIG. 1 which displays asimplified version of the interacting kinase web regulating insulinsensitivity and resistance.

Signal transducing receptors are generally classified into threeclasses. The first class of receptors are receptors that penetrate theplasma membrane and have some intrinsic enzymatic activity.Representative receptors that have intrinsic enzymatic activitiesinclude those that are tyrosine kinases (e.g. PDGF, insulin, EGF and FGFreceptors), tyrosine phosphatases (e.g. CD45 [cluster determinant-45]protein of T cells and macrophages), guanylate cyclases (e.g.natriuretic peptide receptors) and serine/threonine kinases (e.g.activin and TGF-β receptors). Receptors with intrinsic tyrosine kinaseactivity are capable of autophosphorylation as well as phosphorylationof other substrates.

Receptors of the second class are those that are coupled, inside thecell, to GTP-binding and hydrolyzing proteins (termed G-proteins).Receptors of this class which interact with G-proteins have a structurethat is characterized by 7 transmembrane spanning domains. Thesereceptors are termed serpentine receptors. Examples of this class arethe adrenergic receptors, odorant receptors, and certain hormonereceptors (e.g. glucagon, angiotensin, vasopressin and bradykinin).

The third class of receptors may be described as receptors that arefound intracellularly and, upon ligand binding, migrate to the nucleuswhere the ligand-receptor complex directly affects gene transcription.

The proteins which encode for receptor tyrosine kinases (RTK) containfour major domains, those being: a) a transmembrane domain, b) anextracellular ligand binding domain, c) an intracellular regulatorydomain, and d) an intracellular tyrosine kinase domain. The amino acidsequences of RTKs are highly conserved with those of cAMP-dependentprotein kinase (within the ATP and substrate binding regions). RTKproteins are classified into families based upon structural features intheir extracellular portions which include the cysteine rich domains,immunoglobulin-like domains, cadherin domains, leucine-rich domains,Kringle domains, acidic domains, fibronectin type III repeats, discoidinI-like domains, and EGF-like domains. Based upon the presence of thesevarious extracellular domains the RTKs have been sub-divided into atleast 14 different families.

Many receptors that have intrinsic tyrosine kinase activity uponphosphorylation interact with other proteins of the signaling cascade.These other proteins contain a domain of amino acid sequences that arehomologous to a domain first identified in the c-Src proto-oncogene.These domains are termed SH2 domains.

The interactions of SH2 domain containing proteins with RTKs or receptorassociated tyrosine kinases leads to tyrosine phosphorylation of the SH2containing proteins. The resultant phosphorylation produces analteration (either positively or negatively) in that activity. SeveralSH2 containing proteins that have intrinsic enzymatic activity includephospholipase C-γ (PLC-γ), the proto-oncogene c-Ras associated GTPaseactivating protein (rasGAP), phosphatidylinositol-3-kinase (PI-3K),protein tyrosine phosphatase-1C (PTP1C), as well as members of the Srcfamily of protein tyrosine kinases (PTKs).

Non-receptor protein tyrosine kinases (PTK) by and large couple tocellular receptors that lack enzymatic activity themselves. An exampleof receptor-signaling through protein interaction involves the insulinreceptor (IR). This receptor has intrinsic tyrosine kinase activity butdoes not directly interact, following autophosphorylation, withenzymatically active proteins containing SH2 domains (e.g. PI-3K orPLC-γ). Instead, the principal IR substrate is a protein termed IRS-1.

The receptors for the TGF-β superfamily represent the prototypicalreceptor serine/threonine kinase (RSTK). Multifunctional proteins of theTGF-β superfamily include the activins, inhibins and the bonemorphogenetic proteins (BMPs). These proteins can induce and/or inhibitcellular proliferation or differentiation and regulate migration andadhesion of various cell types. One major effect of TGF-β is aregulation of progression through the cell cycle. Additionally, onenuclear protein involved in the responses of cells to TGF-β is c-Myc,which directly affects the expression of genes harboring Myc-bindingelements. PKA, PKC, and MAP kinases represent three major classes ofnon-receptor serine/threonine kinases.

The relationship between kinase activity and disease states is currentlybeing investigated in many laboratories. Such relationships may beeither causative of the disease itself or intimately related to theexpression and progression of disease associated symptomology.Rheumatoid arthritis, an autoimmune disease, provides one example wherethe relationship between kinases and the disease are currently beinginvestigated.

Autoimmune diseases result from a dysfunction of the immune system inwhich the body produces autoantibodies which attack its own organs,tissues and cells—a process mediated via protein phosphorylation.

Over 80 clinically distinct autoimmune diseases have been identified andcollectively afflict approximately 24 million people in the US.Autoimmune diseases can affect any tissue or organ of the body. Becauseof this variability, they can cause a wide range of symptoms and organinjuries, depending upon the site of autoimmune attack. Althoughtreatments exist for many autoimmune diseases, there are no definitivecures for any of them. Treatments to reduce the severity often haveadverse side effects.

Rheumatoid arthritis (RA) is the most prevalent and best studied of theautoimmune diseases and afflicts about 1% of the population worldwide,and for unknown reasons, like other autoimmune diseases, is increasing.RA is characterized by chronic synovial inflammation resulting inprogressive bone and cartilage destruction of the joints. Cytokines,chemokines, and prostaglandins are key mediators of inflammation and canbe found in abundance both in the joint and blood of patients withactive disease. For example, PGE2 is abundantly present in the synovialfluid of RA patients. Increased PGE2 levels are mediated by theinduction of cyclooxygenase-2 (COX-2) and inducible nitric oxidesynthase (iNOS) at inflamed sites. [See, for example van der Kraan P Mand van den Berg W B. Anabolic and destructive mediators inosteoarthritis. Curr Opin Clin Nutr Metab Care, 3:205-211, 2000; Choy EH S and Panayi G S. Cytokine pathways and joint inflammation inrheumatoid arthritis. N Eng J. Med. 344:907-916, 2001; and Wong B R, etal. Targeting Syk as a treatment for allergic and autoimmune disorders.Expert Opin Investig Drugs 13:743-762, 2004.]

The etiology and pathogenesis of RA in humans is still poorlyunderstood, but is viewed to progress in three phases. The initiationphase where dendritic cells present self antigens to autoreactive Tcells. The T cells activate autoreactive B cells via cytokines resultingin the production of autoantibodies, which in turn form immune complexesin joints. In the effector phase, the immune complexes bind Fcfreceptors on macrophages and mast cells, resulting in release ofcytokines and chemokines, inflammation and pain. In the final phase,cytokines and chemokines activate and recruit synovial fibroblasts,osteoclasts and polymorphonuclear neutrophils that release proteases,acids, and ROS such as O2-, resulting in irreversible cartilage and bonedestruction.

In the collagen-induced RA animal model, the participation of T and Bcells is required to initiate the disease. B cell activation signalsthrough spleen tyrosine kinase (Syl) and phosphoinositide 3-kinase(PI3K) following antigen receptor triggering [Ward S G, Finan P.Isoform-specific phosphoinositide 3-kinase inhibitors as therapeuticagents. Curr Opin Pharmacol. August; 3(4):426-34, (2003)]. After theengagement of antigen receptors on B cells, Syk is phosphorylated onthree tyrosines. Syk is a 72-kDa protein-tyrosine kinase that plays acentral role in coupling immune recognition receptors to multipledownstream signaling pathways. This function is a property of both itscatalytic activity and its ability to participate in interactions witheffector proteins containing S142 domains. Phosphorylation of Tyr-317,-342, and -346 create docking sites for multiple SH2 domain containingproteins. [Hutchcroft, J. E., Harrison, M. L. & Geahlen, R. L. (1992).Association of the 72-kDa protein-tyrosine kinase Ptk72 with the B-cellantigen receptor. J. Biol. Chem. 267: 8613-8619, (1992) and Yamada, T.,Taniguchi, T., Yang, C., Yasue, S., Saito, H. & Yamamura, H. Associationwith B-cell antigen cell antigen receptor with protein-tyrosinekinase-P72(Syk) and activation by engagement of membrane IgM. Eur. J.Biochem. 213: 455459, (1993)].

Syk has been shown to be required for the activation of PI3K in responseto a variety of signals including engagement of the B cell antigenreceptor (BCR) and macrophage or neutrophil Fc receptors. [See Crowley,M. T., et al., J. Exp. Med. 186: 1027-1039, (1997); Raeder, E. M., etal., J. Immunol. 163, 6785-6793, (1999); and Jiang, K., et al., Blood101, 236-244, (2003)]. In B cells, the BCR-stimulated activation of PI3Kcan be accomplished through the phosphorylation of adaptor proteins suchas BCAP, CD19, or Gab1, which creates binding sites for the p85regulatory subunit of PI3K. Signals transmitted by many IgG receptorsrequire the activities of both Syk and PI3K and their recruitment to thesite of the clustered receptor. In neutrophils and monocytes, a directassociation of PI3K with phosphorylated immunoreceptor tyrosine basedactivation motif sequences on FcgRIIA was proposed as a mechanism forthe recruitment of PI3K to the receptor. And recently a direct molecularinteraction between Syk and PI3K has been reported [Moon K D, et al.,Molecular Basis for a Direct Interaction between the SykProtein-tyrosine Kinase and Phosphoinositide 3-Kinase. J. Biol. Chem.280, No. 2, Issue of January 14, pp. 1543-1551, (2005)].

Much research has shown that inhibitors of COX-2 activity result indecreased production of PGE2 and are effective in pain relief forpatients with chronic arthritic conditions such as RA. However, concernhas been raised over the adverse effects of agents that inhibit COXenzyme activity since both COX-1 and COX-2 are involved in importantmaintenance functions in tissues such as the gastrointestinal andcardiovascular systems. Therefore, designing a safe, long term treatmentapproach for pain relief in these patients is necessary. Since inducersof COX-2 and iNOS synthesis signal through the Syk, PI3K, p38, ERK1/2,and NF-kB dependent pathways, inhibitors of these pathways may betherapeutic in autoimmune conditions and in particular in the inflamedand degenerating joints of RA patients.

The hops derivative Rho isoalpha acid (RIAA) was found in a screen forinhibition of PGE2 in a RAW 264.7 mouse macrophages model ofinflammation. In the present study, we investigated whether RIAA is adirect COX enzyme inhibitor and/or whether it inhibits the induction ofCOX-2 and iNOS. Our finding that RIAA does not directly inhibit COXenzyme activity, but instead inhibits NF-kB driven enzyme induction leadus to investigate whether RIAA is a kinase inhibitor. Our finding thatRIAA inhibits both Syk and PI3K lead us to test its efficacy in a pilotstudy in patients suffering from various autoimmunine diseases.

Other kinases currently being investigated for their association withdisease symptomology include Aurora, FGFB, MSK, RSE, and SYK.

Aurora—Important regulators of cell division, the Aurora family ofserine/threonine kinases includes Aurora A, B and C. Aurora A and Bkinases have been identified to have direct but distinct roles inmitosis. Over-expression of these three isoforms have been linked to adiverse range of human tumor types, including leukemia, colorectal,breast, prostate, pancreatic, melanoma and cervical cancers.

Fibroblast growth factor receptor (FGFR) is a receptor tyrosine kinase.Mutations in this receptor can result in constitutive activation throughreceptor dimerization, kinase activation, and increased affinity forFGF. FGFR has been implicated in achondroplasia, angiogenesis, andcongenital diseases.

MSK (mitogen- and stress-activated protein kinase) 1 and MSK2 arekinases activated downstream of either the ERK(extracellular-signal-regulated kinase) ½ or p38 MAPK (mitogen-activatedprotein kinase) pathways in vivo and are required for thephosphorylation of CREB (cAMP response element-binding protein) andhistone H3.

Rse is mostly highly expressed in the brain. Rse, also known as Brt,BYK, Dtk, Etk3, Sky, Tif, or sea-related receptor tyrosine kinase, is areceptor tyrosine kinase whose primary role is to protect neurons fromapoptosis. Rse, Axl, and Mer belong to a newly identified family of celladhesion molecule-related receptor tyrosine kinases. GAS6 is a ligandfor the tyrosine kinase receptors Rse, Axl, and Mer. GAS6 functions as aphysiologic anti-inflammatory agent produced by resting EC and depletedwhen pro-inflammatory stimuli turn on the pro-adhesive machinery of EC.

Glycogen synthase kinase-3 (GSK-3), present in two isoforms, has beenidentified as an enzyme involved in the control of glycogen metabolism,and may act as a regulator of cell proliferation and cell death. Unlikemany serine-threonine protein kinases, GSK-3 is constitutively activeand becomes inhibited in response to insulin or growth factors. Its rolein the insulin stimulation of muscle glycogen synthesis makes it anattractive target for therapeutic intervention in diabetes and metabolicsyndrome.

GSK-3 dysregulation has been shown to be a focal point in thedevelopment of insulin resistance. Inhibition of GSK3 improves insulinresistance not only by an increase of glucose disposal rate but also byinhibition of gluconeogenic genes such as phosphoenolpyruvatecarboxykinase and glucose-6-phosphatase in hepatocytes. Furthermore,selective GSK3 inhibitors potentiate insulin-dependent activation ofglucose transport and utilization in muscle in vitro and in vivo. GSK3also directly phosphorylates serine/threonine residues of insulinreceptor substrate-1, which leads to impairment of insulin signaling.GSK3 plays an important role in the insulin signaling pathway and itphosphorylates and inhibits glycogen synthase in the absence of insulin[Parker, P. J., Caudwell, F. B., and Cohen, P. (1983) Eur. J. Biochem.130:227-234]. Increasing evidence supports a negative role of GSK-3 inthe regulation of skeletal muscle glucose transport activity. Forexample, acute treatment of insulin-resistant rodents with selectiveGSK-3 inhibitors improves whole-body insulin sensitivity and insulinaction on muscle glucose transport. Chronic treatment ofinsulin-resistant, pre-diabetic obese Zucker rats with a specific GSK-3inhibitor enhances oral glucose tolerance and whole-body insulinsensitivity, and is associated with an amelioration of dyslipidemia andan improvement in IRS-1-dependent insulin signaling in skeletal muscle.These results provide evidence that selective targeting of GSK-3 inmuscle may be an effective intervention for the treatment ofobesity-associated insulin resistance.

Syk is a non-receptor tyrosine kinase related to ZAP-70 involved insignaling from the B-cell receptor and the IgE receptor. Syk binds toITAM motifs within these receptors, and initiates signaling through theRas, PI 3-kinase, and PLCg signaling pathways. Syk plays a critical rolein intracellular signaling and thus is an important target forinflammatory diseases and respiratory disorders.

Therefore, it would be useful to identify methods and compositions thatwould modulate the expression or activity of single or multiple selectedkinases. The realization of the complexity of the relationship andinteraction among and between the various protein kinases and kinasepathways reinforces the pressing need for developing pharmaceuticalagents capable of acting as protein kinase modulators, regulators orinhibitors that have beneficial activity on multiple kinases or multiplekinase pathways. A single agent approach that specifically targets onekinase or one kinase pathway may be inadequate to treat very complexdiseases, conditions and disorders, such as, for example, diabetes andmetabolic syndrome. Modulating the activity of multiple kinases mayadditionally generate synergistic therapeutic effects not obtainablethrough single kinase modulation.

Such modulation and use may require continual use for chronic conditionsor intermittent use, as needed for example in inflammation, either as acondition unto itself or as an integral component of many diseases andconditions. Additionally, compositions that act as modulators of kinasecan affect a wide variety of disorders in a mammalian body. The instantinvention describes compounds and extracts derived from hops or Acaciawhich may be used to regulate kinase activity, thereby providing a meansto treat numerous disease related symptoms with a concomitant increasein the quality of life.

SUMMARY OF THE INVENTION

The present invention relates generally to methods and compositions formodulating disease associated protein kinases in cells or mammals inneed. In some instances the mammal in need has a condition selected fromthe group consisting of autoimmune disorders, allergic or inflammatorydisorders (including bone), insulin resistance associated disordersincluding aging, cardiovascular disease, lipid storage disorders,metabolic syndrome or diabetes associated disorders, cancer, oculardisorders, and neurological disorders. More specifically, the inventionrelates to methods and compositions comprising extracts, derivatives orfractions isolated either from hops or from members of the plant genusAcacia, or combinations thereof. The invention further relates tomethods and compositions to inhibit inflammatory mediator compounds suchas COX-2 or prostaglandins selectively, or to inhibit inflammatoryresponses through protein kinase modulation in selected target cells.Additionally described are methods and compositions for the treatingsymptomology associated with diseases or conditions selected from thegroup consisting of autoimmune disorders, allergic or inflammatorydisorders (including bone), insulin resistance associated disordersincluding aging, cardiovascular disease, lipid storage disorders,metabolic syndrome or diabetes associated disorders, cancer, oculardisorders, and neurological disorders.

A first embodiment of the invention describes methods for modulating theactivity of a plurality of disease associated protein kinases in asubject in need thereof, wherein said protein kinase modulation isbeneficial to the health of the subject. In this embodiment the methodcomprises administering to the subject-in need a therapeuticallyeffective amount of a composition comprising a compound selected fromthe group consisting of alpha acids, beta acids, prenylflavonoids,chalcones, isoalpha acids, reduced isoalpha acids, spent hops, and acompound or extract derived from acacia.

A second embodiment of the invention describes a method for modulatingthe activity of a plurality of metabolic syndrome associated proteinkinases in a subject in need where the protein kinase modulation isbeneficial to the health of the subject. This method comprisesadministering to the subject in need a therapeutically effective amountof a composition comprising a 5:1 ratio of rho-isoalpha acids to Acacianilotica heartwood or bark powder extract.

A further embodiment of the invention describes compositions formodulating the activity of a plurality of disease associated proteinkinases in a subject in need thereof, where the protein kinasemodulation is beneficial to the health of the subject. In thisembodiment the composition comprises a therapeutically effective amountof a composition comprising a compound selected from the groupconsisting of alpha acids, beta acids, prenylflavonoids, chalcones,isoalpha acids, reduced isoalpha acids, spent hops, and a compound orextract derived from acacia.

Another embodiment of the invention describes a composition formodulating the activity of a plurality of metabolic syndrome associatedprotein kinases in a subject in need where the protein kinase modulationis beneficial to the health of the subject. This composition comprises atherapeutically effective amount of a composition comprising a 5:1 ratioof rho-isoalpha acids to Acacia nilotica heartwood or bark powderextract.

In a further embodiment compositions for modulating the activity of aplurality of ocular disorder associated protein kinases in a subject,wherein said protein kinase modulation is beneficial to the health ofthe subject are described. Here the compositions comprise atherapeutically effective amount of a composition comprising from about1 mg to about 1000 mg of vitamin C; from about 1 IU to about 1000 IU ofvitamin E; from about 0.1 mg to about 2.5 mg of selenium; from about 1mg to about 50 mg of zinc; from about 0.1 mg to about 10 mg of copper;from about 1 mg to about 15 mg of lutein; from about 0.05 mg to about 1mg of zeaxanthin; and from about 1 mg to about 1000 mg of Acacianilotica heartwood powder or bark extract.

Another embodiment of the invention discloses compositions formodulating the activity of a plurality of cancer associated proteinkinases in a subject in need where the protein kinase modulation isbeneficial to the health of the subject. In this embodiment thecomposition comprises a therapeutically effective amount of at least onemember selected from the group consisting of:

-   -   a. from about 0.01 mg to about 10,000 mg of xanthohumol;    -   b. from about 0.01 mg to about 10,000 mg of tetrahydro isoalpha        acid (THIAA);    -   c. from about 0.01 mg to about 10,000 mg of hexahydro isoalpha        acid (HHIAA);    -   d. from about 0.01 mg to about 10,000 mg of RIAA;    -   e. from about 0.01 mg to about 10,000 mg of isoalpha acid (IAA);    -   f. from about 0.01 mg to about 10,000 mg of beta acids; and    -   g. from about 0.01 mg to about 10,000 mg of alpha acids.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 graphically depicts a portion of the kinase network regulatinginsulin sensitivity and resistance.

FIG. 2 graphically depicts the inhibition of five selected kinases byMgRIAA (mgRho).

FIG. 3 graphically depicts the inhibition of PI3K isoforms by five hopscomponents and a Acacia nilotica extract.

FIG. 4 depicts RIAA [panel A] and IAA [panel B] dose-related inhibitionof PGE₂ biosynthesis when added before LPS stimulation of COX-2expression (white bars) or following overnight LPS-stimulation prior tothe addition of test material (grey bars).

FIG. 5 provides a graphic representation of direct enzymatic inhibitionof celecoxib [panel A] and MgRIAA [panel B] on LPS induced COX-2mediated PGE₂ production analyzed in RAW 264.7 cells. PGE₂ was measuredand expressed in pg/ml. The error bars represent the standard deviation(n=8).

FIG. 6 provides Western blot detection of COX-2 protein expression. RAW264.7 cells were stimulated with LPS for the indicated times, afterwhich total cell extract was visualized by western blot for COX-2 andGAPDH expression [panel A]. Densitometry of the COX-2 and GAPDH bandswas performed. The graph [panel B] represents the ratio of COX-2 toGAPDH.

FIG. 7 provides Western blot detection of iNOS protein expression. RAW264.7 cells were stimulated with LPS for the indicated times, afterwhich total cell extract was visualized by western blot for iNOS andGAPDH expression [panel A]. Densitometry of the iNOS and GAPDH bands wasperformed. The graph [panel B] represents the ratio of iNOS to GAPDH.

FIG. 8 provides a representative schematic of the TransAM NF-κB kitutilizing a 96-well format. The oligonucleotide bound to the platecontains the consensus binding site for NF-κB. The primary antibodydetected the p50 subunit of NF-κB.

FIG. 9 provides representative binding activity of NF-κB as determinedby the TransAM NF-κB kit. The percent of DNA binding was calculatedrelative to the LPS control (100%). The error bars represent thestandard deviation (n=2). RAW 264.7 cells were treated with testcompounds and LPS for 4 hr as described in the Examples section.

FIG. 10 is a schematic of a representative testing procedure forassessing the lipogenic effect of an Acacia sample #4909 extract ondeveloping and mature adipocytes. The 3T3-L1 murine fibroblast model wasused to study the potential effects of the test compounds on adipocyteadipogenesis.

FIG. 11 is a graphic representation depicting the nonpolar lipid contentof 3T3-L1 adipocytes treated with an Acacia sample #4909 extract or thepositive controls indomethacin and troglitazone relative to the solventcontrol. Error bars represent the 95% confidence limits (one-tail).

FIG. 12 is a schematic of a representative testing procedure forassessing the effect of a dimethyl sulfoxide-soluble fraction of anaqueous extract of Acacia sample #4909 on the secretion of adiponectinfrom insulin-resistant 3T3-L1 adipocytes.

FIG. 13 is a representative bar graph depicting maximum adiponectinsecretion by insulin-resistant 3T3-L1 cells in 24 hr elicited by threedoses of troglitazone and four doses of a dimethyl sulfoxide-solublefraction of an aqueous extract of Acacia sample #4909. Values presentedare percent relative to the solvent control; error bars represent 95%confidence intervals.

FIG. 14 is a schematic of a representative testing protocol forassessing the effect of a dimethyl sulfoxide-soluble fraction of anaqueous extract of Acacia sample #4909 on the secretion of adiponectinfrom 3T3-L1 adipocytes treated with test material plus 10, 2 or 0.5 ngTNFα/ml.

FIG. 15 depicts representative bar graphs representing adiponectinsecretion by TNFα treated mature 3T3-L1 cells elicited by indomethacinor an Acacia sample #4909 extract. Values presented are percent relativeto the solvent control; error bars represent 95% confidence intervals.*Significantly different from TNFα alone treatment (p<0.05).

FIG. 16 graphically illustrates the relative increase in triglyceridecontent in insulin resistant 3T3-L1 adipocytes by various compositionsof Acacia catechu and A. nilotica from different commercial sources.Values presented are percent relative to the solvent control; error barsrepresent 95% confidence intervals.

FIG. 17 graphically depicts a representation of the maximum relativeadiponectin secretion elicited by various extracts of Acacia catechu.Values presented are percent relative to the solvent control; error barsrepresent 95% confidence intervals.

FIG. 18 graphically depicts the lipid content (relative to the solventcontrol) of 3T3-L1 adipocytes treated with hops compounds or thepositive controls indomethacin and troglitazone. The 3T3-L1 murinefibroblast model was used to study the potential effects of the testcompounds on adipocyte adipogenesis. Results are represented as relativenonpolar lipid content of control cells; error bars represent the 95%confidence interval.

FIG. 19 is a representative bar graph of maximum adiponectin secretionby insulin-resistant 3T3-L1 cells in 24 hr elicited by the test materialover four doses. Values presented are as a percent relative to thesolvent control; error bars represent 95% confidence intervals.IAA=isoalpha acids, RIAA=Rho isoalpha acids, HHIA=hexahydroisoalphaacids, and THIAA=tetrahydroisoalpha acids.

FIG. 20 depicts the Hofstee plots for Rho isoalpha acids, isoalphaacids, tetrahydroisoalpha acids, hexahydroisoalpha acids, xanthohumols,spent hops, hexahydrocolupulone and the positive control troglitazone.Maximum adiponectin secretion relative to the solvent control wasestimated from the y-intercept, while the concentration of test materialnecessary for half maximal adiponectin secretion was commuted from thenegative value of the slope.

FIG. 21 displays two bar graphs representing relative adiponectinsecretion by TNFα-treated, mature 3T3-L1 cells elicited by isoalphaacids and Rho isoalpha acids [panel A], and hexahydro isoalpha acids andtetrahydro isoalpba acids [panel B]. Values presented are percentrelative to the solvent control; error bars represent 95% confidenceintervals. *Significantly different from TNFα only treatment (p<0.05).

FIG. 22 depicts NF-kB nuclear translocation in insulin-resistant 3T3-L1adipocytes [panel A] three and [panel B] 24 hr following addition of 10ng TNFα/ml. Pioglitazone, RIAA and xanthohumols were added at 5.0 (blackbars) and 2.5 (stripped bars) μg/ml. Jurkat nuclear extracts from cellscultured in medium supplemented with 50 ng/ml TPA (phorbol,12-myristate, 13 acetate) and 0.5 μM calcium ionophore A23187 (CI) fortwo hours at 37° C. immediately prior to harvesting.

FIG. 23 graphically describes the relative triglyceride content ofinsulin resistant 3T3-L1 cells treated with solvent, metformin, anAcacia sample #5659 aqueous extract or a 1:1 combination ofmetformin/Acacia catechu extract. Results are represented as a relativetriglyceride content of fully differentiated cells in the solventcontrols.

FIG. 24 graphically depicts the effects of 10 μg/ml of solvent control(DMSO), RIAA, isoalpha acid (IAA), tetrahydroisoalpha acid (THIAA), a1:1 mixture of THIAA and hexahydroisoalpha acid (HHIAA), xanthohumol(XN), LY 249002 (LY), ethanol (ETOH), alpha acid (ALPHA), and beta acid(BETA) on cell proliferation in the RL 95-2 endometrial cell line.

FIG. 25 graphically depicts the effects of various concentrations ofTHIAA or reduced isoalpha acids (RIAA) on cell proliferation in theHT-29 cell line.

FIG. 26 graphically depicts the effects of various concentrations ofTHIAA or reduced isoalpha acids (RIAA) on cell proliferation in theSW480 cell line.

FIG. 27 graphically depicts the dose responses of various combinationsof reduced isoalpha acids (RIAA) and Acacia for reducing serum glucose[panel A] and serum insulin [panel B] in the db/db mouse model.

FIG. 28 graphically depicts the reduction in serum glucose [panel A] andserum insulin [panel. B] in the db/db mouse model produced by a 5:1combination of RIAA:Acacia as compared to the pharmaceuticalanti-diabetic compounds roziglitazone and metformin.

FIG. 29 graphically depicts the effects of reduced isoalpha acids (RIAA)on the arthritic index in a murine model of rheumatoid arthritis.

FIG. 30 graphically depicts the effects of THIAA on the arthritic indexin a murine model of rheumatoid arthritis.

FIG. 31 graphically depicts the effects of RIAA/Acacia (1:5)supplementation (3 tablets per day) on fasting and 2 h post-prandial(pp) insulin levels. For the 2 h pp insulin level assessment, subjectspresented after a 10-12 h fast and consumed a solution containing 75 gglucose (Trutol 100, CASCO NERL® Diagnostics); 2 h after the glucosechallenge, blood was drawn and assayed for insulin levels (LaboratoriesNorthwest, Tacoma, Wash.).

FIG. 32 graphically depicts the effects of RIAA/Acacia (1:5)supplementation (3 tablets per day) on fasting and 2 h pp glucoselevels. For the 2 h pp glucose level assessment, subjects presentedafter a 10-12 h fast and consumed a solution containing 75 g glucose(Trutol 100, CASCO NERL® Diagnostics); 2 h after the glucose challenge,blood was drawn and assayed for glucose levels (Laboratories Northwest,Tacoma, Wash.).

FIG. 33 graphically depicts the effects of RIAA/Acacia (1:5)supplementation (3 tablets per day) on HOMA scores. HOMA score wascalculated from fasting insulin and glucose by published methods[(insulin (mcRIU/mL)*glucose (mg/dL))/405].

FIG. 34 graphically depicts the effects of RIAA/Acacia (1:5)supplementation (3 tablets per day) on serum TG levels.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods and compositions for modulatingprotein kinases in cells or mammals in need. In some instances themammal in need has a condition selected from the group consisting ofautoimmune disorders, allergic or inflammatory disorders, metabolicsyndrome or diabetes associated disorders, cancer, and neurologicaldisorders. More specifically, the invention relates to a compositions,methods and kits comprising extracts, derivatives or fractions isolatedeither from hops or from members of the plant genus Acacia, orcombinations thereof. The invention further relates to compositions andmethods to inhibit inflammatory mediators such as cyclooxygenase-2(COX-2) or prostaglandins selectively, or to inhibit inflammatoryresponses through protein kinase modulation in selected target cells.

The patents, published applications, and scientific literature referredto herein establish the knowledge of those with skill in the art and arehereby incorporated by reference in their entirety to the same extent asif each was specifically and individually indicated to be incorporatedby reference. Any conflict between any reference cited herein and thespecific teachings of this specification shall be resolved in favor ofthe latter. Likewise, any conflict between an art-understood definitionof a word or phrase and a definition of the word or phrase asspecifically taught in this specification shall be resolved in favor ofthe latter.

Technical and scientific terms used herein have the meaning commonlyunderstood by one of skill in the art to which the present inventionpertains, unless otherwise defined. Reference is made herein to variousmethodologies and materials known to those of skill in the art. Standardreference works setting forth the general principles of recombinant DNAtechnology include Sambrook et al., Molecular Cloning: A LaboratoryManual, 2nd Ed., Cold Spring Harbor Laboratory Press, New York (1989);Kaufman et al., Eds., Handbook of Molecular and Cellular Methods inBiology in Medicine, CRC Press, Boca Raton (1995); McPherson, Ed.,Directed Mutagenesis: A Practical Approach, IRL Press, Oxford (1991).Standard reference works setting forth the general principles ofpharmacology include Goodman and Gilman's The Pharmacological Basis ofTherapeutics, 11th Ed., McGraw Hill Companies Inc., New York (2006).

In the specification and the appended claims, the singular forms includeplural referents unless the context clearly dictates otherwise. As usedin this specification, the singular forms “a,” “an” and “the”specifically also encompass the plural forms of the terms to which theyrefer, unless the content clearly dictates otherwise. Additionally, asused herein, unless specifically indicated otherwise, the word “or” isused in the “inclusive” sense of “and/or” and not the “exclusive” senseof “either/or.” The term “about” is used herein to mean approximately,in the region of, roughly, or around. When the term “about” is used inconjunction with a numerical range, it modifies that range by extendingthe boundaries above and below the numerical values set forth. Ingeneral, the term “about” is used herein to modify a numerical valueabove and below the stated value by a variance of 20%.

As used herein, the recitation of a numerical range for a variable isintended to convey that the invention may be practiced with the variableequal to any of the values within that range. Thus, for a variable whichis inherently discrete, the variable can be equal to any integer valueof the numerical range, including the end-points of the range.Similarly, for a variable which is inherently continuous, the variablecan be equal to any real value of the numerical range, including theend-points of the range. As an example, a variable which is described ashaving values between 0 and 2, can be 0, 1 or 2 for variables which areinherently discrete, and can be 0.0, 0.1, 0.01, 0.001, or any other realvalue for variables which are inherently continuous.

Reference is made hereinafter in detail to specific embodiments of theinvention. While the invention will be described in conjunction withthese specific embodiments, it will be understood that it is notintended to limit the invention to such specific embodiments. On thecontrary, it is intended to cover alternatives, modifications, andequivalents as may be included within the spirit and scope of theinvention as defined by the appended claims. In the followingdescription, numerous specific details are set forth in order to providea thorough understanding of the present invention. The present inventionmay be practiced without some or all of these specific details. In otherinstances, well known process operations have not been described indetail, in order not to unnecessarily obscure the present invention.

Any suitable materials and/or methods known to those of skill can beutilized in carrying out the present invention. However, preferredmaterials and methods are described. Materials, reagents and the like towhich reference are made in the following description and examples areobtainable from commercial sources, unless otherwise noted.

A first embodiment of the invention discloses methods for modulating theactivity of a plurality of disease associated protein kinases in asubject in need thereof, where the protein kinase modulation isbeneficial to the health of the subject. Methods of this embodimentcomprise administering to the subject in need a therapeuticallyeffective amount of a composition comprising a compound selected fromthe group consisting of alpha acids, beta acids, prenylflavonoids,chalcones, isoalpha acids, reduced isoalpha acids, spent hops, and acompound or extract derived from acacia.

In some aspects of this embodiment the subject in need has a conditionselected from the group consisting of autoimmune disorders, allergic orinflammatory disorders, metabolic syndrome or diabetes associateddisorders, cancer, ocular disorders, and neurological disorders, whilein other aspects the disease associated protein kinase is selected fromthe group consisting of ABL, AKT, AURORA, CDK, DBF2/20, EGFR,EPH/ELK/ECK, ERK/MAPKFGFR, GSK3, IKKB, INSR, JAK DOM 1/2, MARK/PRKAA,MEK/STE7, MEKK/STE11, MLK, mTOR, PAK/STE20, PDGFR, PI3K, PKC, POLO, SRC,TEC/ATK, and ZAP/SYK.

In still other aspects the alpha acid is selected from the groupconsisting of humulone, cohumulone, adhumulone, prehumulone, andposthumulone or the beta acid is selected from the group consisting oflupulone, colupulone, adlupulone, and prelupulone.

In yet other aspects the isoalpha acid of the composition used isselected from the group consisting of isohumulone, isoadhumulone, andisocohumulone. While the compositions employ RIAA, the RIAA is selectedfrom the group consisting of dihydro-isohumulone, dihydro-isocohumulone,dihydro-adhumulone, tetrahydro-isohumulone, tetrahydro-isocohumulone,tetrahydro-adhumulone, hexahydro-isohumulone, hexahydro-isocohumulone,and hexahydro-adhumulone. The rho-isoalpha acid configuration isemployed in yet other aspects of this embodiment.

In some aspects of this embodiment the chalcone is xanthohumol orisoxanthohumol, or the prenylflavonoid is 6-prenylnaringenin or8-prenylnaringenin.

In yet other aspects, the compound or extract derived from acacia isderived from Acacia catechu or Acacia nilotica. In those aspects wherethe acacia derived compound or extract is derived from Acacia catechu orAcacia nilotica, the Acacia catechu or Acacia nilotica compound isselected from the group consisting of gum resin, bark powder, heartwoodpowder, and an Acacia catechu or Acacia nilotica extract. In thoseaspects where the acacia derived compound is an Acacia catechu or Acacianilotica extract, the extract is selected from acidic, alkaline, polarsolvent, nonpolar solvent, and gastric fluid extracts.

Compositions used in the methods of this embodiment may further compriseone or more members selected from the group consisting of antioxidants,vitamins, minerals, proteins, fats, and carbohydrates, or apharmaceutically acceptable excipient selected from the group consistingof coatings, isotonic and absorption delaying agents, binders,adhesives, lubricants, disintergrants, coloring agents, flavoringagents, sweetening agents, absorbants, detergents, and emulsifyingagents.

In some methods of this embodiment, the composition used furthercomprises an antidiabetic drug selected from the group consisting ofrosiglitazone, troglitazone, pioglitazone, and metformin.

As used herein, “disease associated kinase” means those individualprotein kinases or groups or families of kinases that are eitherdirectly causative of the disease or whose activation is associated withpathways which serve to exacerbate the symptoms of the disease inquestion. Furthermore, as used herein “disease”, “condition”, or“disorder” may be used interchangeably.

The phrase “protein kinase modulation is beneficial to the health of thesubject” refers to those instances wherein the kinase modulation (eitherup or down regulation) results in reducing, preventing, and/or reversingthe symptoms of the disease or augments the activity of a secondarytreatment modality.

As used in this specification, whether in a transitional phrase or inthe body of the claim, the terms “comprise(s)” and “comprising” are tobe interpreted as having an open-ended meaning. That is, the terms areto be interpreted synonymously with the phrases “having at least” or“including at least”. When used in the context of a process, the term“comprising” means that the process includes at least the recited steps,but may include additional steps. When used in the context of a compoundor composition, the term “comprising” means that the compound orcomposition includes at least the recited features or compounds, but mayalso include additional features or compounds.

As used herein, the terms “derivatives” or a matter “derived” refer to achemical substance related structurally to another substance andtheoretically obtainable from it, i.e. a substance that can be made fromanother substance. Derivatives can include compounds obtained via achemical reaction.

As used herein, the term “hop extract” refers to the solid materialresulting from (1) exposing a hops plant product to a solvent, (2)separating the solvent from the hops plant products, and (3) eliminatingthe solvent. “Spent hops” refers to the hops plant products remainingfollowing a hops extraction procedure. See Verzele, M. and DeKeukeleire, D., Developments in Food Science 27; Chemistry and Analysisof Hop and Beer Bitter Acids, Elsevier Science Pub. Co., 1991, New York,USA, herein incorporated by reference in its entirety, for a detaileddiscussion of hops chemistry. As used herein when in reference to aRIAA, “Rho” refers to those reduced isoalpha acids wherein the reductionis a reduction of the carbonyl group in the 4-methyl-3-pentenoyl sidechain.

As used herein, the term “solvent” refers to a liquid of aqueous ororganic nature possessing the necessary characteristics to extract solidmaterial from the hop plant product. Examples of solvents would include,but not limited to, water, steam, superheated water, methanol, ethanol,hexane, chloroform, liquid CO₂, liquid N₂ or any combinations of suchmaterials.

As used herein, the term “CO₂ extract” refers to the solid materialresulting from exposing a hops plant product to a liquid orsupercritical CO₂ preparation followed by subsequent removal of the CO₂.

The term “pharmaceutically acceptable” is used in the sense of beingcompatible with the other ingredients of the compositions and notdeleterious to the recipient thereof.

As used herein, “compounds” may be identified either by their chemicalstructure, chemical name, or common name. When the chemical structureand chemical or common name conflict, the chemical structure isdeterminative of the identity of the compound. The compounds describedherein may contain one or more chiral centers and/or double bonds andtherefore, may exist as stereoisomers, such as double-bond isomers(i.e., geometric isomers), enantiomers or diastereomers. Accordingly,the chemical structures depicted herein encompass all possibleenantiomers and stereoisomers of the illustrated or identified compoundsincluding the stereoisomerically pure form (e.g., geometrically pure,enantiomerically pure or diastereomerically pure) and enantiomeric andstereoisomeric mixtures. Enantiomeric and stereoisomeric mixtures can beresolved into their component enantiomers or stereoisomers usingseparation techniques or chiral synthesis techniques well known to theskilled artisan. The compounds may also exist in several tautomericforms including the enol form, the keto form and mixtures thereof.Accordingly, the chemical structures depicted herein encompass allpossible tautomeric forms of the illustrated or identified compounds.The compounds described also encompass isotopically labeled compoundswhere one or more atoms have an atomic mass different from the atomicmass conventionally found in nature. Examples of isotopes that may beincorporated into the compounds of the invention include, but are notlimited to, ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, etc. Compounds may exist inunsolvated forms as well as solvated forms, including hydrated forms andas N-oxides. In general, compounds may be hydrated, solvated orN-oxides. Certain compounds may exist in multiple crystalline oramorphous forms. Also contemplated within the scope of the invention arecongeners, analogs, hydrolysis products, metabolites and precursor orprodrugs of the compound. In general, unless otherwise indicated, allphysical forms are equivalent for the uses contemplated herein and areintended to be within the scope of the present invention.

Compounds according to the invention may be present as salts. Inparticular, pharmaceutically acceptable salts of the compounds arecontemplated. A “pharmaceutically acceptable salt” of the invention is acombination of a compound of the invention and either an acid or a basethat forms a salt (such as, for example, the magnesium salt, denotedherein as “Mg” or “Mag”) with the compound and is tolerated by a subjectunder therapeutic conditions. In general, a pharmaceutically acceptablesalt of a compound of the invention will have a therapeutic index (theratio of the lowest toxic dose to the lowest therapeutically effectivedose) of 1 or greater. The person skilled in the art will recognize thatthe lowest therapeutically effective dose will vary from subject tosubject and from indication to indication, and will thus adjustaccordingly.

As used herein “hop” or “hops” refers to plant cones of the genusHumulus which contain a bitter aromatic oil which is used in the brewingindustry to prevent bacterial action and add the characteristic bittertaste to beer. More preferably, the hops used are derived from Humuluslupulus.

The term “acacia”, as used herein, refers to any member of leguminoustrees and shrubs of the genus Acacia. Preferably, the botanical compoundderived from acacia is derived from Acacia catechu or Acacia nilotica.

The compounds according to the invention are optionally formulated in apharmaceutically acceptable vehicle with any of the well knownpharmaceutically acceptable carriers, including diluents and excipients(see Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, MackPublishing Co., Easton, Pa. 1990 and Remington: The Science and Practiceof Pharmacy, Lippincott, Williams & Wilkins, 1995). While the type ofpharmaceutically acceptable carrier/vehicle employed in generating thecompositions of the invention will vary depending upon the mode ofadministration of the composition to a mammal, generallypharmaceutically acceptable carriers are physiologically inert andnon-toxic. Formulations of compositions according to the invention maycontain more than one type of compound of the invention), as well anyother pharmacologically active ingredient useful for the treatment ofthe symptom/condition being treated.

In some aspects of this embodiment, the modulated kinase is selectedfrom the group consisting of Abl, Abl(T315I), ALK, ALK4, AMPK, Arg, Arg,ARK5, ASK1, Aurora-A, Axl, Blk, Bmx, BRK, BrSK1, BrSK2, BTK, CaMKI,CaMKII, CaMKIV, CDK1/cyclinB, CDK2/cyclinA, CDK2/cyclinE, CDK3/cyclinE,CDK5/p25, CDK5/p35, CDK6/cyclinD3, CDK7/cyclinH/MAT1, CDK9/cyclin T1,CHK1, CHK2, CK1(y), CK1δ, CK2, CK2α2, cKit(D816V), cKit, c-RAF, CSK,cSRC, DAPK1, DAPK2, DDR2, DMPK, DRAK1, DYRK2, EGFR, EGFR(L858R),EGFR(L861Q), EphA1, EphA2, EphA3, EphA4, EphA5, EphA7, EplA8, EphB1,EphB2, EphB3, EphB4, ErbB4, Fer, Fes, FGFR1, FGFR2, FGFR3, FGFR4, Fgr,Flt1, Flt3 (D835Y), Flt3, Flt4, Fms, Fyn, GSK3β, GSK3α, Hck, HIPK1,HIPK2, HIPK3, IGF-1R, IKKB, IKKA, IR, IRAK1, IRAK4, IRR, ITK, JAK2,JAK3, JNK1α1, JNK2α2, JNK3, KDR, Lck, LIMK1, LKB1, LOK, Lyn, Lyn, MAPK1,MAPK2, MAPK2, MAPKAP-K2, MAPKAP-K3, MARK1, MEK1, MELK, Met, MINK, MKK4,MKK6, MKK71, MLCK, MLK1, Mnk2, NMCK13, MRCKα, MSK1, MSK2, MSSK1, MST1,MST2, MST3, MuSK, NEK2, NEK3, NEK6, NEK7, NLK, p70S6K, PAK2, PAK3,PAK-4, PAK6, PAR-1Bα, PDGFRβ, PDGFRα, PDK1, PI3K beta, PI3K delta, PI3Kgamma, Pim-1, Pim-2, PKA(b), PKA, PKBβ, PKBα, PKBγ, PKCμ, PKCβI, PKCβII,PKCα, PKCγ, PKCδ, PKCε, PKCζ, PKCη, PKCθ, PKCι, PKD2, PKG1β, PKG1α,Plk3, PRAK, PRK2, PrKX, PTK5, Pyk2, Ret, RIPK2, ROCK-I, ROCK-II,ROCK-II, Ron, Ros, Rse, Rsk1, Rsk1, Rsk2, Rsk3, SAPK2a, SAPK2a(T106M),SAPK2b, SAPK3, SAPK4, SGK, SGK2, SGK3, SIK, Snk, SRPK1, SRPK2, STK33,Syk, TAK1, TBK1, Tie2, TrkA, TrkB, TSSK1, TSSK2, WNK2, WNK3, Yes,ZAP-70, and ZIPK.

In preferred aspects the modulated kinase is selected from the groupconsisting of Aurora-A, CDK2/cyclin A, DAPK1, DAPK2, EphA3FGFR4, GSK3β,GSK3α, Hck, MAPK1, MAPKAP-K2, MSK2, MSSK1, PI3K beta, PI3K delta, PI3Kgamma, Rse, Rsk2, Syk, and Tie2. In more preferred aspects the modulatedkinase is selected from the group consisting of ABL, AKT, AURORA, CDK,DBF2/20, EGFR, EPH/ELK/ECK, ERK/MAPKFGFR, GSK3, IKKB, INSR, JAK DOM 1/2,MARK/PRKAA, MEK/STE7, MEKK/STE11, MLK, mTOR, PAK/STE20, PDGFR, PI3K,PKC, POLO, SRC, TEC/ATK, and ZAP/SYK.

In some aspects, the mammal in need has a condition selected from thegroup consisting of autoimmune disorders, allergic or inflammatorydisorders, metabolic syndrome or diabetes associated disorders, cancer,ocular disorders, and neurological disorders. In those aspects where themammal in need has an autoimmune disorder, that disorder is selectedfrom the group consisting of autoimmune hemolytic anemia, Crohn'sdisease, inflammatory bowel disease, multiple sclerosis, myastheniagravis, rheumatoid arthritis, and systemic lupus erythematosus.

In other aspects of this embodiment, the allergic or inflammatorydisorder is selected from the group consisting of asthma, rhinitis,ulcerative colitis, Crohn's disease, pancreatitis, gastritis, benigntumors, polyps, hereditary polyposis syndrome, colon cancer, rectalcancer, breast cancer, prostate cancer, stomach cancer, ulcerous diseaseof the digestive organs, stenocardia, atherosclerosis, myocardialinfarction, sequelae of stenocardia or myocardial infarction, seniledementia, inflammation associated with microbial infection (e.g.,fungal, bacterial, or viral), and cerebrovascular diseases. In yet otheraspects the metabolic syndrome or diabetes associated disorder isselected from the group consisting of metabolic syndrome, diabetes type1, diabetes type 2, insulin insensitivity and obesity.

In some aspects the cancer is selected from the group consisting ofbrain, breast, colon, kidney, leukemia, liver, lung, and prostatecancers. In yet other aspects of this embodiment the ocular disorder isselected from retinopathy, diabetic retinopathy, and maculardegeneration. In still other aspects of this embodiment, theneurological disorder is selected from the group consisting ofAlzheimer's disease, Parkinson's disease, multiple sclerosis,amyotrophic lateral sclerosis (ALS or Lou Gehrig's Disease),Huntington's disease, neurocognitive dysfunction, senile dementia, andmood disorder diseases.

The term “modulate” or “modulation” is used herein to mean the up ordown regulation of expression or activity of the enzyme by a compound,ingredient, etc., to which it refers.

As used herein, the term “protein kinase” represent transferase classenzymes that are able to transfer a phosphate group from a donormolecule to an amino acid residue of a protein. See Kostich, M., et al.,Human Members of the Eukaryotic Protein Kinase Family, Genome Biology3(9):research0043.1-0043.12, 2002 herein incorporated by reference inits entirety, for a detailed discussion of protein kinases andfamily/group nomenclature.

Representative, non-limiting examples of kinases include Abl,Abl(T315I), ALK, ALK4, AMPK, Arg, Arg, ARK5, ASK1, Aurora-A, Axl, Blk,Bmx, BRK, BrSK1, BrSK2, BTK, CaMKI, CaMKII, CaMKIV, CDK1/cyclinB,CDK2/cyclinA, CDK2/cyclinE, CDK3/cyclinE, CDK5/p25, CDK5/p35,CDK6/cyclinD3, CDK7/cyclinH/MAT1, CDK9/cyclin T1, CHK1, CHK2, CK1(y),CK1δ, CK2, CK2α2, cKit(D816V), cKit, c-RAF, CSK, cSRC, DAPK1, DAPK2,DDR2, DMPK, DRAK1, DYRK2, EGFR, EGFR(L858R), EGFR(L861Q), EphA1, EphA2,EphA3, EphA4, EphA5, EphA7, EphA8, EphB1, EphB2, EphB3, EphB4, ErbB4,Fer, Fes, FGFR1, FGFR2, FGFR3, FGFR4, Fgr, Flt1, Flt3(D835Y), Flt3,F1t4, Fms, Fyn, GSK313, GSK3α, Hck, HIPK1, HIPK2, HIPK3, IGF-1R, IKKβ,IKKα, IR, IRAK1, IRAK4, IRR, ITK, JAK2, JAK3, JNK1α1, JNK2α2, JNK3, KDR,Lck, LIMK1, LKB1, LOK, Lyn, Lyn, MAPK1, MAPK2, MAPK2, MAPKAP-K2,MAPKAP-K3, MARK1, MEK1, MELK, Met, MINK, MKK4, MKK6, MKK713, MLCK, MLK1,Mnk2, MRCKβ, MRCKα, MSK1, MSK2, MSSK1, MST1, MST2, MST3, MuSK, NEK2,NEK3, NEK6, NEK7, NLK, p70S6K, PAK2, PAK3, PAK-4, PAK6, PAR-1Bα,PDGFR13, PDGFRα, PDK1, PI3K beta, PI3K delta, PI3K gamma, Pim-1, Pim-2,PKA(b), PKA, PKBβ, PKBα, PKBγ, PKCμ, PKCβI, PKCβII, PKCα, PKCγ, PKCδ,PKCε, PKCζ, PKCη, PKCθ, PKCι, PKD2, PKG1β, PKG1α, Plk3, PRAK, PRK2,PrKX, PTK5, Pyk2, Ret, RIPK2, ROCK-I, ROCK-II, ROCK-II, Ron, Ros, Rse,Rsk1, Rsk1, Rsk2, Rsk3, SAPK2a, SAPK2a(T106M), SAPK2b, SAPK3, SAPK4,SGK, SGK2, SGK3, SIK, Snk, SRPK1, SRPK2, STK33, Syk, TAK1, TBK1, Tie2,TrkA, TrkB, TSSK1, TSSK2, WNK2, WNK3, Yes, ZAP-70, ZIPK. In someembodiments, the kinases may be ALK, Aurora-A, Axl, CDK9/cyclin T1,DAPK1, DAPK2, Fer, FGFR4, GSK31, GSK3c, Hck, JNK2α2, MSK2, p70S6K, PAK3,PI3K delta, PI3K gamma, PKA, PKBβ, PKBα, Rse, Rsk2, Syk, TrkA, andTSSK1. In yet other embodiments the kinase is selected from the groupconsisting of ABL, AKT, AURORA, CDK, DBF2/20, EGFR, EPH/ELK/ECK,ERK/MAPKFGFR, GSK3, IKKB, INSR, JAK DOM 1/2, MARK/PRKAA, MEK/STE7,MEKK/STE11, MLK, mTOR, PAK/STE20, PDGFR, PI3K, PKC, POLO, SRC, TEC/ATK,and ZAP/SYK.

The methods and compositions of the present invention are intended foruse with any mammal that may experience the benefits of the methods ofthe invention. Foremost among such mammals are humans, although theinvention is not intended to be so limited, and is applicable toveterinary uses. Thus, in accordance with the invention, “mammals” or“mammal in need” include humans as well as non-human mammals,particularly domesticated animals including, without limitation, cats,dogs, and horses.

As used herein, “autoimmune disorder” refers to those diseases,illnesses, or conditions engendered when the host's systems are attackedby the host's own immune system. Representative, non-limiting examplesof autoimmune diseases include alopecia greata, ankylosing spondylitis,arthritis, antiphospholipid syndrome, autoimmune Addison's disease,autoimmune hemolytic anemia, autoimmune inner ear disease (also known asMeniers disease), autoimmune lymphoproliferative syndrome (ALPS),autoimmune thrombocytopenic purpura, autoimmune hemolytic anemia,autoimmune hepatitis, Bechet's disease, Crohn's disease, diabetesmellitus type 1, glomerulonephritis, Graves' disease, Guillain-Barrésyndrome, inflammatory bowel disease, lupus nephritis, multiplesclerosis, myasthenia gravis, pemphigus, pernicious anemia,polyarteritis nodosa, polymyositis, primary billiary cirrhosis,psoriasis, rheumatic fever, rheumatoid arthritis, scleroderma, Sjögren'ssyndrome, systemic lupus erythematosus, ulcerative colitis, vitiligo,and Wegener's granulamatosis. Representative, non-limiting examples ofkinases associated with autoimmune disorders include BTK, ERK, FGFR,FMS, GSK, IGFR, IKK, JAK, PDGFR, PI3K, PKC, PLK, ROCK, and VEGFR.

“Allergic disorders”, as used herein, refers to an exaggerated orpathological reaction (as by sneezing, respiratory distress, itching, orskin rashes) to substances, situations, or physical states that arewithout comparable effect on the average individual. As used herein,“inflammatory disorders” means a response (usually local) to cellularinjury that is marked by capillary dilatation, leukocytic infiltration,redness, heat, pain, swelling, and often loss of function and thatserves as a mechanism initiating the elimination of noxious agents andof damaged tissue. Examples of allergic or inflammatory disordersinclude, without limitation, asthma, rhinitis, ulcerative colitis,Crohn's disease, pancreatitis, gastritis, benign tumors, polyps,hereditary polyposis syndrome, colon cancer, rectal cancer, breastcancer, prostate cancer, stomach cancer, ulcerous disease of thedigestive organs, stenocardia, atherosclerosis, myocardial infarction,sequelae of stenocardia or myocardial infarction, senile dementia, andcerebrovascular diseases. Representative, non-limiting examples ofkinases associated with allergic disorders include AKT, BTK, EGFR, FYN,IKKB, ITK, JAK, KIT, LCK, LYN, MAPK, MEK, mTOR, PDGFR, PI3K, PKC, PPAR,ROCK, SRC, SYK, and ZAP.

As used herein, “metabolic syndrome” and “diabetes associated disorders”refers to insulin related disorders, i.e., to those diseases orconditions where the response to insulin is either causative of thedisease or has been implicated in the progression or suppression of thedisease or condition. Representative examples of insulin relateddisorders include, without limitation diabetes, diabetic complications,insulin sensitivity, polycystic ovary disease, hyperglycemia,dyslipidemia, insulin resistance, metabolic syndrome, obesity, bodyweight gain, inflammatory diseases, diseases of the digestive organs,stenocardia, myocardial infarction, sequelae of stenocardia ormyocardial infarction, senile dementia, and cerebrovascular dementia.See, Harrison's Principles of Internal Medicine, 16 h Ed., McGraw HillCompanies Inc., New York (2005). Examples, without limitation, ofinflammatory conditions include diseases of the digestive organs (suchas ulcerative colitis, Crohn's disease, pancreatitis, gastritis, benigntumor of the digestive organs, digestive polyps, hereditary polyposissyndrome, colon cancer, rectal cancer, stomach cancer and ulcerousdiseases of the digestive organs), stenocardia, myocardial infarction,sequelae of stenocardia or myocardial infarction, senile dementia,cerebrovascular dementia, immunological diseases and cancer in general.Non-limiting examples of kinases associated with metabolic syndrome caninclude AKT, AMPK, CDK, ERK, GSK, IGFR, JNK, MAPK, MEK, PI3K, and PKC.

“Insulin resistance” refers to a reduced sensitivity to insulin by thebody's insulin-dependent processes resulting in lowered activity ofthese processes or an increase in insulin production or both. Insulinresistance is typical of type 2 diabetes but may also occur in theabsence of diabetes.

As used herein “diabetic complications” include, without limitation,retinopathy, muscle infarction, idiopathic skeletal hyperostosis andbone loss, foot ulcers, neuropathy, arteriosclerosis, respiratoryautonomic neuropathy and structural derangement of the thorax and lungparenchyma, left ventricular hypertrophy, cardiovascular morbidity,progressive loss of kidney function, and anemia.

As used herein “cancer” refers to any of various benign or malignantneoplasms characterized by the proliferation of anaplastic cells that,if malignant, tend to invade surrounding tissue and metastasize to newbody sites. Representative, non-limiting examples of cancers consideredwithin the scope of this invention include brain, breast, colon, kidney,leukemia, liver, lung, and prostate cancers. Non-limiting examples ofcancer associated protein kinases considered within the scope of thisinvention include ABL, AKT, Aurora, BRK, CDK, EGFR, ERB, FGFR, IGFR,KIT, MAPK, mTOR, PDGFR, PI3K, PKC, and SRC.

“Ocular disorders”, refers to those disturbances in the structure orfunction of the eye resulting from developmental abnormality, disease,injury, age or toxin. Non-limiting examples of ocular disordersconsidered within the scope of the present invention includeretinopathy, macular degeneration or diabetic retinopathy. Oculardisorder associated kinases include, without limitation, Aurora, EPH,ERB, ERK, FMS, IGFR, MEK, PDGFR, PI3K, PKC, SRC, and VEGFR.

A “neurological disorder”, as used herein, refers to any disturbance inthe structure or function of the central nervous system resulting fromdevelopmental abnormality, disease, injury or toxin. Representative,non-limiting examples of neurological disorders include Alzheimer'sdisease, Parkinson's disease, multiple sclerosis, amyotrophic lateralsclerosis (ALS or Lou Gehrig's Disease), Huntington's disease,neurocognitive dysfunction, senile dementia, and mood disorder diseases.Protein kinases associated with neurological disorders may include,without limitation, CDK, FYN, JNK, MAPK, PKC, ROCK, RTK, SRC, and VEGFR.

As used herein “cardiovascular disease” or “CVD” refers to thosepathologies or conditions which impair the function of, or destroycardiac tissue or blood vessels. Cardiovascular disease associatedkinases include, without limitation, AKT, GRK, GSK, IKKB, JAK, JUN,MAPK, PKC, RHO, ROCK, and TOR.

“Osteoporosis”, as used herein, refers to a disease in which the boneshave become extremely porous, thereby making the bone more susceptibleto fracture and slower healing. Protein kinases associated withosteoporosis include, without limitation, AKT, CAMK, IRAK-M, MAPK, mTOR,PPAR, RHO, ROS, SRC, SYR, and VEGFR.

An embodiment of the invention describes methods modulating the activityof a plurality of metabolic syndrome associated protein kinases in asubject in need thereof, wherein the protein kinase modulation isbeneficial to the health of the subject. This method comprisesadministering to the subject in need a therapeutically effective amountof a composition comprising a 5:1 (w/w) ratio of rho-isoalpha acids toAcacia nilotica heartwood powder extract.

A further embodiment of the invention discloses composition formodulating the activity of a plurality of disease associated proteinkinases in a subject in need thereof, where the protein kinasemodulation is beneficial to the health of the subject. Compositions ofthis embodiment comprise a therapeutically effective amount of acompound selected from the group consisting of alpha acids, beta acids,prenylflavonoids, chalcones, isoalpha acids, reduced isoalpha acids,spent hops, and a compound or extract derived from acacia.

In some aspects of this embodiment the subject in need has a conditionselected from the group consisting of autoimmune disorders, allergic orinflammatory disorders, metabolic syndrome or diabetes associateddisorders, cancer, ocular disorders, and neurological disorders, whilein other aspects the disease associated protein kinase is selected fromthe group consisting of ABL, AKT, AURORA, CDK, DBF2/20, EGFR,EPH/ELK/ECK, ERK/MAPKFGFR, GSK3, IKKB, INSR, JAK DOM 1/2, MARK/PRKAA,MEK/STE7, MEKK/STE11, MLK, mTOR, PAK/STE20, PDGFR, PI3K, PKC, POLO, SRC,TEC/ATK, and ZAP/SYK.

In still other aspects the alpha acid is selected from the groupconsisting of humulone, cohumulone, adhumulone, prehumulone, andposthumulone or the beta acid is selected from the group consisting oflupulone, colupulone, adlupulone, and prelupulone.

In yet other aspects the isoalpha acid of the composition used isselected from the group consisting of isohumulone, isoadhumulone, andisocohumulone. While the compositions employ RIAA, the RIAA is selectedfrom the group consisting of dihydro-isohumulone, dihydro-isocohumulone,dihydro-adhumulone, tetrahydro-isohumulone, tetrahydro-isocohumulone,tetrahydro-adhumulone, hexahydro-isohumulone, hexahydro-isocohumulone,and hexahydro-adhumulone. The rho-isoalpha acid configuration isemployed in yet other aspects of this embodiment.

In some aspects of this embodiment the chalcone is xanthohumol orisoxanthohumol, or the prenylflavonoid is 6-prenylnaringenin or8-prenylnaringenin.

In yet other aspects, the compound or extract derived from acacia isderived from Acacia catechu or Acacia nilotica. In those aspects wherethe acacia derived compound or extract is derived from Acacia catechu orAcacia nilotica, the Acacia catechu or Acacia nilotica compound isselected from the group consisting of gum resin, bark powder, heartwoodpowder, and an Acacia catechu or Acacia nilotica extract. In thoseaspects where the acacia derived compound is an Acacia catechu or Acacianilotica extract, the extract is selected from acidic, alkaline, polarsolvent, nonpolar solvent, and gastric fluid extracts, Compositions ofthis embodiment may further comprise one or more members selected fromthe group consisting of antioxidants, vitamins, minerals, proteins,fats, and carbohydrates, or a pharmaceutically acceptable excipientselected from the group consisting of coatings, isotonic and absorptiondelaying agents, binders, adhesives, lubricants, disintergrants,coloring agents, flavoring agents, sweetening agents, absorbants,detergents, and emulsifying agents.

In some aspects of this embodiment, the composition used furthercomprises an antidiabetic drug selected from the group consisting ofrosiglitazone, troglitazone, pioglitazone, and metformin.

In some aspects of this embodiment, the modulated kinase is selectedfrom the group consisting of Abl, Abl(T315I), ALK, ALK4, AMPK, Arg, Arg,ARK5, ASK1, Aurora-A, Axl, Blk, Bmx, BRK, BrSK1, BrSK2, BTK, CaMKI,CaMKII, CaMKIV, CDK1/cyclinB, CDK2/cyclinA, CDK2/cyclinE, CDK3/cyclinE,CDK5/p25, CDK5/p35, CDK6/cyclinD3, CDK7/cyclin/MAT1, CDK9/cyclin T1,CHK1, CHK2, CK1(y), CK1δ, CK2, CK2α2, cKit(D816V), cKit, c-RAF, CSK,cSRC, DAPK1, DAPK2, DDR2, DMPK, DRAK1, DYRK2, EGFR, EGFR(L858R),EGFR(L861Q), EphA1, EphA2, EphA3, EphA4, EphA5, EphA7, EphA8, EphB1,EphB2, EphB3, EphB4, ErbB4, Fer, Fes, FGFR1, FGFR2, FGFR3, FGFR4, Fgr,Flt1, Flt3(D835Y), Flt3, Flt4, Fms, Fyn, GSK3β, GSK3α, Hck, HIPK1,HIPK2, HIPK3, IGF-1R, IKKβ, IKKα, IR, IRAK1, IRAK4, IRR, ITK, JAK2,JAK3, JN1α1, JNK2α2, JNK3, KDR, Lck, LIMK1, LKB1, LOK, Lyn, Lyn, MAPK1,MAPK2, MAPK2, MAPKAP-K2, MAPKAP-K3, MARK1, MEK1, MELK, Met, MINK, MKK4,MKK6, MKK7J3, MLCK, MLK1, Mnk2, MRCKβ, MRCKα, MSK1, MSK2, MSSK1, MST1,MST2, MST3, MuSK, NEK2, NEK3, NEK6, NEK7, NLK, p70S6K, PAK2, PAK3,PAK-4, PAK6, PAR-1Bα, PDGFRβ, PDGFRα, PDK1, PI3K beta, PI3K delta, PI3Kgamma, Pim-1, Pim-2, PKA(b), PKA, PKBβ, PKBα, PKBγ, PKCμ, PKCβI, PKCβII,PKCα, PKCγ, PKCδ, PKCε, PKCζ, PKCη, PKCθ, PKCι, PKD2, PKG1β, PKG1α,Plk3, PRAK, PRK2, PrKX, PTK5, Pyk2, Ret, RIPK2, ROCK-I, ROCK-II,ROCK-II, Ron, Ros, Rse, Rsk1, Rsk1, Rsk2, Rsk3, SAPK2a, SAPK2a(T106M),SAPK2b, SAPK3, SAPK4, SGK, SGK2, SGK3, SIK, Snk, SRPK1, SRPK2, STK33,Syk, TAK1, TBK1, Tie2, TrkA, TrkB, TSSK1, TSSK2, WNK2, WNK3, Yes,ZAP-70, and ZIPK.

In preferred aspects the modulated kinase is selected from the groupconsisting of Aurora-A, CDK2/cyclin A, DAPK1, DAPK2, EphA3FGFR4, GSK313,GSK3a, Hck, MAPK1, MAPKAP-K2, MSK2, MSSK1, PI3K beta, PI3K delta, PI3Kgamma, Rse, Rsk2, Syk, and Tie2. In more preferred aspects the modulatedkinase is selected from the group consisting of ABL, AKT, AURORA, CDK,DBF2/20, EGFR, EPH/ELK/ECK, ERK/MAPKFGFR, GSK3, IKKB, INSR, JAK DOM ½,MARK/PRKAA, MEK/STE7, MEKK/STE11, MLK, mTOR, PAK/STE20, PDGFR, PI3K,PKC, POLO, SRC, TEC/ATK, and ZAP/SYK.

In some aspects, the mammal in need has a condition selected from thegroup consisting of autoimmune disorders, allergic or inflammatorydisorders, metabolic syndrome or diabetes associated disorders, cancer,ocular disorders, and neurological disorders. In those aspects where themammal in need has an autoimmune disorder, that disorder is selectedfrom the group consisting of autoimmune hemolytic anemia, Crohn'sdisease, inflammatory bowel disease, multiple sclerosis, myastheniagravis, rheumatoid arthritis, and systemic lupus erythematosus.

In other aspects of this embodiment, the allergic or inflammatorydisorder is selected from the group consisting of asthma, rhinitis,ulcerative colitis, Crohn's disease, pancreatitis, gastritis, benigntumors, polyps, hereditary polyposis syndrome, colon cancer, rectalcancer, breast cancer, prostate cancer, stomach cancer, ulcerous diseaseof the digestive organs, stenocardia, atherosclerosis, myocardialinfarction, sequelae of stenocardia or myocardial infarction, seniledementia, and cerebrovascular diseases. In yet other aspects themetabolic syndrome or diabetes associated disorder is selected from thegroup consisting of metabolic syndrome, diabetes type 1, diabetes type2, insulin insensitivity and obesity.

In some aspects the cancer is selected from the group consisting ofbrain, breast, colon, kidney, leukemia, liver, lung, and prostatecancers. In yet other aspects of this embodiment the ocular disorder isselected from retinopathy, diabetic retinopathy, and maculardegeneration. In still other aspects of this embodiment, theneurological disorder is selected from the group consisting ofAlzheimer's disease, Parkinson's disease, multiple sclerosis,amyotrophic lateral sclerosis (ALS or Lou Gehrig's Disease),Huntington's disease, neurocognitive dysfunction, senile dementia, andmood disorder diseases.

Another embodiment of the invention describes compositions formodulating the activity of a plurality of metabolic syndrome associatedprotein kinases in a subject in need thereof, wherein the protein kinasemodulation is beneficial to the health of the subject. Thesecompositions comprise a therapeutically effective amount of acomposition comprising a 5:1 (w/w) ratio of rho-isoalpha acids to Acacianilotica heartwood powder extract.

Another embodiment of the invention discloses compositions formodulating the activity of a plurality of cancer associated proteinkinases in a subject in need where the protein kinase modulation isbeneficial to the health of the subject. In this embodiment thecomposition comprises a therapeutically effective amount of at least onemember selected from the group consisting of:

-   -   a. from about 0.01 mg to about 10,000 mg of xanthohumol;    -   b. from about 0.01 mg to about 10,000 mg of THIAA;    -   c. from about 0.01 mg to about 10,000 mg of HHIAA;    -   d. from about 0.01 mg to about 10,000 mg of RIAA;    -   e. from about 0.01 mg to about 10,000 mg of IAA;    -   f. from about 0.01 mg to about 10,000 mg of beta acids; and    -   g. from about 0.01 mg to about 10,000 mg of alpha acids.

As used herein, by “treating” is meant reducing, preventing, and/orreversing the symptoms in the individual to which a compound of theinvention has been administered, as compared to the symptoms of anindividual not being treated according to the invention. A practitionerwill appreciate that the compounds, compositions, and methods describedherein are to be used in concomitance with continuous clinicalevaluations by a skilled practitioner (physician or veterinarian) todetermine subsequent therapy. Hence, following treatment thepractitioners will evaluate any improvement in the treatment of thepulmonary inflammation according to standard methodologies. Suchevaluation will aid and inform in evaluating whether to increase, reduceor continue a particular treatment dose, mode of administration, etc.

It will be understood that the subject to which a compound of theinvention is administered need not suffer from a specific traumaticstate. Indeed, the compounds of the invention may be administeredprophylactically, prior to any development of symptoms. The term“therapeutic,” “therapeutically,” and permutations of these terms areused to encompass therapeutic, palliative as well as prophylactic uses.Hence, as used herein, by “treating or alleviating the symptoms” ismeant reducing, preventing, and/or reversing the symptoms of theindividual to which a compound of the invention has been administered,as compared to the symptoms of an individual receiving no suchadministration.

The term “therapeutically effective amount” is used to denote treatmentsat dosages effective to achieve the therapeutic result sought.Furthermore, one of skill will appreciate that the therapeuticallyeffective amount of the compound of the invention may be lowered orincreased by fine tuning and/or by administering more than one compoundof the invention, or by administering a compound of the invention withanother compound. See, for example, Meiner, C. L., “Clinical Trials:Design, Conduct, and Analysis,” Monographs in Epidemiology andBiostatistics, Vol. 8 Oxford University Press, USA (1986). The inventiontherefore provides a method to tailor the administration/treatment tothe particular exigencies specific to a given mammal. As illustrated inthe following examples, therapeutically effective amounts may be easilydetermined for example empirically by starting at relatively low amountsand by step-wise increments with concurrent evaluation of beneficialeffect.

It will be appreciated by those of skill in the art that the number ofadministrations of the compounds according to the invention will varyfrom patient to patient based on the particular medical status of thatpatient at any given time including other clinical factors such as age,weight and condition of the mammal and the route of administrationchosen.

As used herein, “symptom” denotes any sensation or change in bodilyfunction that is experienced by a patient and is associated with aparticular disease, i.e., anything that accompanies “X” and is regardedas an indication of “X”'s existence. It is recognized and understoodthat symptoms will vary from disease to disease or condition tocondition. By way of non-limiting examples, symptoms associated withautoimmune disorders include fatigue, dizziness, malaise, increase insize of an organ or tissue (for example, thyroid enlargement in Grave'sDisease), or destruction of an organ or tissue resulting in decreasedfunctioning of an organ or tissue (for example, the islet cells of thepancreas are destroyed in diabetes).

Representative symptomology for allergy associated diseases orconditions include absentmindedness, anaphylaxis, asthma, burning eyes,constipation, coughing, dark circles under or around the eyes,dermatitis, depression, diarrhea, difficulty swallowing, distraction ordifficulty with concentration, dizziness, eczema, embarrassment,fatigue, flushing, headaches, heart palpitations, hives, impaired senseof smell, irritability/behavioral problems, itchy nose or skin orthroat, joint aches muscle pains, nasal congestion, nasal polyps,nausea, postnasal drainage (postnasal drip), rapid pulse, rhinorrhea(runny nose), ringing—popping or fullness in the ears, shortness ofbreath, skin rashes, sleep difficulties, sneezing, swelling(angioedema), throat hoarseness, tingling nose, tiredness, vertigo,vomiting, watery or itchy or crusty or red eyes, and wheezing.

“Inflammation” or “inflammatory condition” as used herein refers to alocal response to cellular injury that is marked by capillarydilatation, leukocytic infiltration, redness, heat, pain, swelling, andoften loss of function and that serves as a mechanism initiating theelimination of noxious agents and of damaged tissue. Representativesymptoms of inflammation or an inflammatory condition include, ifconfined to a joint, redness, swollen joint that's warm to touch, jointpain and stiffness, and loss of joint function. Systemic inflammatoryresponses can produce “flu-like” symptoms, such as, for instance, fever,chills, fatigue/loss of energy, headaches, loss of appetite, and musclestiffness.

Diabetes and metabolic syndrome often go undiagnosed because many oftheir symptoms seem so harmless. For example, some diabetes symptomsinclude, without limitation: frequent urination, excessive thirst,extreme hunger, unusual weight loss, increased fatigue, irritability,and blurry vision.

Symptomology of neurological disorders may be variable and can include,without limitation, numbness, tingling, hyperesthesia (increasedsensitivity), paralysis, localized weakness, dysarthria (difficultspeech), aphasia (inability to speak), dysphagia (difficultyswallowing), diplopia (double vision), cognition issues (inability toconcentrate, for example), memory loss, amaurosis fugax (temporary lossof vision in one eye) difficulty walking, incoordination, tremor,seizures, confusion, lethargy, dementia, delirium and coma.

In a further embodiment compositions for modulating the activity of aplurality of ocular disorder associated protein kinases in a subject,wherein said protein kinase modulation is beneficial to the health ofthe subject are described. Here the compositions comprise atherapeutically effective amount of a composition comprising from about1 mg to about 1000 mg of vitamin C; from about 1 IU to about 1000 IU ofvitamin E; from about 0.1 mg to about 2.5 mg of selenium; from about 1mg to about 50 mg of zinc; from about 0.1 mg to about 10 mg of copper;from about 1 mg to about 15 mg of lutein; from about 0.05 mg to about 1mg of zeaxanthin; and from about 1 mg to about 1000 mg of Acacianilotica heartwood powder extract

The following examples are intended to further illustrate certainpreferred embodiments of the invention and are not limiting in nature.Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, numerous equivalents to thespecific substances and procedures described herein.

EXAMPLES Example 1 Effects of Modified Hops Components on ProteinKinases

As stated above, kinases represent transferase class enzymes that areable to transfer a phosphate group from a donor molecule (usually ATP)to an amino acid residue of a protein (usually threonine, serine ortyrosine). Kinases are used in signal transduction for the regulation ofenzymes, i.e., they can inhibit or activate the activity of an enzyme,such as in cholesterol biosynthesis, amino acid transformations, orglycogen turnover. While most kinases are specialized to a single kindof amino acid residue, some kinases exhibit dual activity in that theycan phosphorylate two different kinds of amino acids. As shown in FIG.1, kinases function in signal transduction and translation.

Methods—The inhibitory effect of 10 μg RIAA/ml of the present inventionon human kinase activity was tested on a panel of over 200 kinases inthe KinaseProfiler™ Assay (Upstate Cell Signaling Solutions, UpstateUSA, Inc., Charlottesville, Va., USA). The assay protocols for specifickinases are summarized athttp://www.upstate.com/img/pdf/kp_protocols_full.pdf (last visited onJun. 12, 2006).

Results—Just over 205 human kinases were assayed in the cell freesystem. Surprisingly we discovered that the hops compounds testedinhibited 25 of the 205 kinases by 10% or greater. Eight (8) of the 205were inhibited by >20%; 5 of 205 were inhibited by >30; and 2 wereinhibited by about 50%.

Specifically in the PI3kinase pathway, hops inhibits PI3Kγ, PI3Kδ,PI3Kβ, Akt1, Akt2, GSK3a, GSK3p, P70S6K. It should be noted that mTORwas not available for testing.

The inhibitory effects of the hops compounds RIAA on the kinases testedare shown in Table 1 below.

TABLE 1 Kinase inhibition by RIAA tested in the KinaseProfiler ™ Assayat 10 μg/ml Kinase % of Control Abl 93 Abl 102 Abl(T315I) 121 ALK 84ALK4 109 AMPK 103 Arg 96 Arg 95 ARK5 103 ASK1 116 Aurora-A 77 Axl 89 Blk115 Bmx 108 BRK 112 BrSK1 108 BrSK2 100 BTK 97 CaMKI 96 CaMKII 119CaMKIV 115 CDK1/cyclinB 109 CDK2/cyclinA 94 CDK2/cyclinE 122CDK3/cyclinE 104 CDK5/p25 100 CDK5/p35 103 CDK6/cyclinD3 110CDK7/cyclinH/MAT1 108 CDK9/cyclin T1 84 CHK1 102 CHK2 98 CK1(y) 109 CK1δ104 CK2 122 CK2α2 126 cKit(D816V) 135 cKit 103 c-RAF 101 CSK 108 cSRC103 DAPK1 78 DAPK2 67 DDR2 108 DMPK 121 DRAK1 111 DYRK2 112 EGFR 120EGFR(L858R) 113 EGFR(L861Q) 122 EphA1 105 EphA2 115 EphA3 93 EphA4 108EphA5 120 EphA7 127 EphA8 112 EphB1 134 EphB2 110 EphB3 101 EphB4 113ErbB4 123 Fer 80 Fes 121 FGFR1 96 FGFR2 103 FGFR3 109 FGFR4 83 Fgr 102Flt1 102 Flt3(D835Y) 103 Flt3 108 Flt4 110 Fms 105 Fyn 100 GSK3β 82GSK3α 89 Hck 83 HIPK1 98 HIPK2 113 HIPK3 119 IGF-1R 97 IKKβ 117 IKKα 117IR 95 IRAK1 109 IRAK4 110 IRR 102 ITK 117 JAK2 112 JAK3 111 JNK1α1 104JNK2α2 84 JNK3 98 KDR 101 Lck 94 LIMK1 102 LKB1 106 LOK 127 Lyn 100 Lyn109 MAPK1 95 MAPK2 101 MAPK2 113 MAPKAP-K2 98 MAPKAP-K3 97 MARK1 101MEK1 113 MELK 98 Met 109 MINK 109 MKK4 94 MKK6 114 MKK7β 113 MLCK 114MLK1 109 Mnk2 116 MRCKβ 114 MRCKα 119 MSK1 97 MSK2 89 MSSK1 92 MST1 105MST2 103 MST3 104 MuSK 100 NEK2 99 NEK3 109 NEK6 98 NEK7 98 NLK 109p70S6K 87 PAK2 92 PAK3 54 PAK4 99 PAK6 109 PAR-1Bα 109 PDGFRβ 109 PDGFRα101 PDK1 118 PI3K beta 95 PI3K delta 88 PI3K gamma 80 Pim-1 133 Pim-2112 PKA(b) 99 PKA 66 PKBβ 87 PKBα 49 PKBγ 100 PKCμ 100 PKCβI 112 PKCβII99 PKCα 109 PKCγ 109 PKCδ 101 PKCε 99 PKCζ 107 PKCη 119 PKCθ 117 PKCι 96PKD2 115 PKG1β 99 PKG1α 110 Plk3 98 PRAK 100 PRK2 102 PrKX 94 PTK5 104Pyk2 112 Ret 96 RIPK2 98 ROCK-I 105 ROCK-II 90 ROCK-II 105 Ron 102 Ros94 Rse 84 Rsk1 93 Rsk1 95 Rsk2 89 Rsk3 95 SAPK2a 111 SAPK2a(T106M) 108SAPK2b 100 SAPK3 98 SAPK4 98 SGK 94 SGK2 96 SGK3 107 SIK 90 Snk 98 SRPK1117 SRPK2 110 STK33 94 Syk 82 TAK1 109 TBK1 121 Tie2 95 TrkA 85 TrkB 91TSSK1 51 TSSK2 97 WNK2 102 WNK3 104 Yes 92 ZAP-70 113 ZIPK 91

It should be noted that several kinases in the PI3K pathway are beingpreferentially inhibited by RIAA, for example, Akt1 at 51% inhibition.It is interesting to note that three Akt isoforms exist. Akt1 null miceare viable, but retarded in growth [Cho et al., Science 292:1728-1731(2001)]. Drosophila eye cells deficient in Akt1 are reduced in size[Verdu et al., Nat cell Biol 1:500-505 (1999)]; overexpression leads toincreased size from normal. Akt2 null mice are viable but have impairedglucose control [Cho et al., J Biol Chem 276:38345-38352 (2001)]. Hence,it appears Akt1 plays a role in size determination and Akt2 is involvedin insulin signaling.

The PI3K pathway is known to play a key role in mRNA stability and mRNAtranslation selection resulting in differential protein expression ofvarious oncogene proteins and inflammatory pathway proteins. Aparticular 5′ mRNA structure denoted 5′-TOP has been shown to be a keystructure in the regulation of mRNA translation selection.

A review of the cPLA literature and DNA sequence indicates that the 5′mRNA of human cPLA2 contains a consensus (82% homology to a knownoncogene regulated similarly) sequence indicating that it too has a5′TOP structure. sPLAs, also known to be implicated in inflammation,also have this same 5′-TOP. Moreover, this indicates that cPLA2 andpossibly other PLAs are upregulated by the PI3K pathway via increasingthe translation selection of cPLA2 mRNA resulting in increases in cPLA2protein. Conversely, inhibitors of PI3K should reduce the amount ofcPLA2 and reduce PGE₂ formation made via the COX2 pathway.

Taken together the kinase data and our own results where we havediscovered that hops compounds inhibit cPLA2 protein expression (Westernblots, data not shown) but not mRNA, suggests that the anti-inflammatorymode of action of hops compounds may be via reducing substrateavailability to COX2 by reducing cPLA2 protein levels, and perhaps morespecifically, by inhibiting the PI3K pathway resulting in the inhibitionof activation of TOP mRNA translation.

The exact pathway of activity remains unclear. Some reports areconsistent with the model that activation occurs via phosphorylation ofone or more of the six isoforms of ribosomal protein S6 (RPS6). RPS6 isreported to resolve the 5′TOP mRNA allowing efficient translation intoprotein. However, Stolovich et al. Mol Cell Biol Dec, 8101-8113 (2002),disputes this model and proposes that Akt1 phosphorylates an unknowntranslation factor, X, which allows TOP mRNA translation.

Example 2 Dose Response Effects of Hops or Acacia Components on SelectedProtein Kinases

The dose responsiveness of mgRho was tested at approximately 10, 50, and100 μg/ml on over sixty selected protein kinases according to theprotocols of Example 1 are presented as Table 2 below. The five kinaseswhich were inhibited the most are displayed graphically as FIG. 2.

The dose responsiveness for kinase inhibition (reported as a percent ofcontrol) of a THIAA preparation was tested at approximately 1, 10, 25,and 50 ug/ml on 86 selected kinases as presented in Table 3 below.Similarly, an acacia preparation was tested at approximately 1, 5, and25 ug/ml on over 230 selected protein kinases according to the protocolsof Example 1 and are presented as Table 4 below. Preparations ofisoalpha acids (IAA), heaxahydroisoalpha acids (HHIAA), beta acids, andxanthohumol were also tested at approximately 1, 10, 25, and 50 ug/ml on86 selected kinases and the dose responsiveness results are presentedbelow as Tables 5-8 respectively.

TABLE 2 Dose response effect (as % of Control) of a mgRho on selectedprotein kinases Kinase 10 ug/ml 50 ug/ml 100 ug/ml Abl 103 82 65 ALK 7993 109 AMPK 107 105 110 Arg 94 76 64 Aurora-A 96 59 33 Axl 101 87 85CaMKI 95 85 77 CDK2/cyclinA 106 81 59 CDK9/cyclin T1 100 88 101 c-RAF105 109 103 DAPK1 82 56 51 DAPK2 64 51 45 EphA3 103 64 55 Fer 87 74 83FGFR1 98 99 93 FGFR4 111 68 35 GSK3β 65 17 26 GSK3α 65 64 13 Hck 86 7259 IKKβ 104 91 92 IKKα 104 101 96 IR 87 85 78 JNK1α1 105 115 106 JNK2α2119 136 124 JNK3 98 98 86 Lck 105 83 81 MAPK1 77 53 44 MAPK2 101 104 106MAPKAP-K2 111 99 49 MAPKAP-K3 109 106 73 MEK1 106 104 91 MKK4 110 110 98MSK2 92 54 43 MSSK1 120 31 26 p70S6K 105 86 69 PAK2 99 84 89 PAK5 99 9478 PASK 105 111 102 PDK1 98 90 78 PI3K beta (est) 74 49 39 PI3K delta(est) 64 22 13 PI3K gamma (est) 85 69 55 PKA 103 95 92 PKCε 96 93 91PKCι 100 94 96 PrKX 100 105 90 ROCK-II 102 101 99 Ros 105 86 90 Rse 7139 22 Rsk2 108 79 56 Rsk3 108 102 86 SAPK2a 96 105 109 SAPK2a(T106M) 100107 107 SAPK2b 101 102 106 SAPK3 110 109 110 SAPK4 97 107 109 SGK 111105 94 SIK 130 125 117 STK33 99 96 103 Syk 79 46 28 Tie2 113 74 56 TrkA127 115 93 TrkB 106 105 81 TSSK1 105 100 95 Yes 100 105 100 ZIPK 92 6283

TABLE 3 Dose response effect (as % of Control) of THIAA on selectedprotein kinases Kinase 1 ug/ml 5 ug/ml 25 ug/ml 50 ug/ml Abl(T315I) 10495 68 10 ALK4 127 112 108 AMPK 135 136 139 62 Aurora-A 102 86 50 5 Bmx110 105 57 30 BTK 104 86 58 48 CaMKI 163 132 65 16 CaMKIIβ 106 102 90 71CaMKIIγ 99 101 87 81 CaMKIIδ 99 103 80 76 CaMKIV 99 117 120 126 CaMKIδ91 95 61 43 CDK1/cyclinB 82 101 77 66 CDK2/cyclinA 118 113 87 50CDK2/cyclinE 87 79 73 57 CDK3/cyclinE 113 111 105 32 CDK5/p25 102 100 8554 CDK5/p35 109 106 89 80 CDK6/cyclinD3 114 113 112 70 CDK9/cyclin T1106 93 66 36 CHK1 116 118 149 148 CHK2 111 116 98 68 CK1(y) 101 101 55CK1γ1 101 100 42 43 CK1γ2 94 85 33 48 CK1γ3 99 91 23 18 CK1δ 109 97 6542 cKit(D816H) 113 113 69 75 CSK 110 113 92 137 cSRC 105 103 91 17 DAPK162 34 21 14 DAPK2 60 54 41 17 DRAK1 113 116 75 18 EphA2 110 112 85 31EphA8 110 110 83 43 EphB1 153 177 196 53 ErbB4 124 125 75 56 Fer 85 4124 12 Fes 112 134 116 57 FGFR1 109 110 110 111 FGFR1(V561M) 97 106 91 92FGFR2 126 115 58 7 FGFR3 112 94 39 16 FGFR4 122 93 83 58 Fgr 121 120 11047 Flt4 126 119 85 31 IKKα 139 140 140 102 JNK1α1 71 118 118 107 JNK2α294 97 98 101 JNK3 121 78 58 44 KDR 106 107 104 126 Lck 97 105 125 88LKB1 145 144 140 140 MAPK2 99 109 112 102 Pim-1 103 100 44 44 Pim-2 103109 83 22 PKA(b) 104 77 32 0 PKA 104 101 90 25 PKBβ 117 102 27 33 PKBα103 101 49 50 PKBγ 107 109 99 33 PKCμ 90 90 93 87 PKCβII 99 107 103 64PKCα 110 111 112 102 PKCγ 86 95 77 62 PKCδ 97 93 84 87 PKCε 76 88 88 90PKCζ 93 100 107 103 PKCη 82 99 103 90 PKCθ 93 95 86 90 PKCι 77 90 93 134PRAK 99 81 21 33 PrKX 92 76 32 38 Ron 120 110 97 42 Ros 105 105 94 93Rsk1 101 87 48 31 Rsk2 100 85 40 14 SGK 98 103 79 77 SGK2 117 110 45 18Syk 99 93 55 17 TBK1 101 100 82 56 Tie2 109 115 100 32 TrkA 107 65 30 15TrkB 97 96 72 21 TSSK2 112 111 87 66 ZIPK 106 101 74 59

TABLE 4 Dose response effect (as % of Control) of acacia on selectedprotein kinases Kinase 1 ug/ml 5 ug/ml 25 ug/ml Abl 53 27 2 Abl(T315I)57 26 11 ALK 102 52 10 ALK4 84 96 98 AMPK 108 101 77 Arg 86 53 23 Arg106 55 18 ARK5 36 13 6 ASK1 100 70 23 Aurora-A 8 −1 3 Axl 64 17 4 Blk 31−2 −3 Bmx 101 51 0 BRK 47 19 7 BrSK1 58 6 2 BrSK2 82 16 4 BTK 15 −1 −3CaMKI 97 90 49 CaMKII 83 50 6 CaMKIIβ 87 45 10 CaMKIIγ 90 51 12 CaMKIIδ25 13 6 CaMKIV 89 44 44 CaMKIδ 69 19 10 CDK1/cyclinB 62 48 9CDK2/cyclinA 69 15 5 CDK2/cyclinE 51 14 8 CDK3/cyclinE 41 13 4 CDK5/p2582 41 7 CDK5/p35 77 46 13 CDK6/cyclinD3 100 54 5 CDK7/cyclinH/MAT1 12490 42 CDK9/cyclin T1 79 21 4 CHK1 87 52 17 CHK2 52 16 5 CK1(y) 77 32 3CK1γ1 51 7 −4 CK1γ2 31 5 1 CK1γ3 49 16 0 CK1δ 60 15 6 CK2 157 162 128CK2α2 95 83 51 cKit(D816H) 27 7 2 cKit(D816V) 111 91 41 cKit 94 68 24cKit(V560G) 49 5 0 cKit(V654A) 30 8 3 CLK3 33 16 6 c-RAF 105 100 87 CSK74 19 1 cSRC 99 12 0 DAPK1 90 72 12 DAPK2 75 31 4 DCAMKL2 107 106 77DDR2 84 91 45 DMPK 105 106 116 DRAK1 92 40 11 DYRK2 83 55 25 eEF-2K 10397 59 EGFR 76 26 6 EGFR(L858R) 99 40 1 EGFR(L861Q) 90 49 1 EGFR(T790M)93 29 7 EGFR(T790M, L858R) 74 30 4 EphA1 106 43 9 EphA2 94 82 6 EphA3 9483 50 EphA4 55 12 6 EphA5 100 28 10 EphA7 103 80 6 EphA8 113 84 19 EphB1116 63 8 EphB2 30 5 2 EphB3 109 35 1 EphB4 30 11 3 ErbB4 61 8 0 FAK 10678 2 Fer 106 134 28 Fes 143 74 43 FGFR1 125 26 3 FGFR1(V561M) 92 50 2FGFR2 73 −2 −5 FGFR3 21 3 1 FGFR4 30 7 5 Fgr 78 18 7 Flt1 41 12 1Flt3(D835Y) 65 15 −1 Flt3 76 16 3 Flt4 12 3 2 Fms 94 73 19 Fyn 23 5 1GRK5 96 91 81 GRK6 117 117 94 GSK3β 13 5 4 GSK3α 5 2 1 Hck 87 29 −2HIPK1 110 112 62 HIPK2 92 71 24 HIPK3 106 92 56 IGF-1R 148 122 41 IKKβ30 6 3 IKKα 120 86 11 IR 121 123 129 IRAK1 98 85 49 IRAK4 117 95 47 IRR91 70 28 Itk 121 114 48 JAK2 83 69 23 JAK3 24 7 1 JNK1α1 118 110 75JNK2α2 99 106 102 JNK3 52 23 3 KDR 90 60 18 Lck 92 93 25 LIMK1 108 10453 LKB1 126 122 98 LOK 103 72 27 Lyn 4 1 2 MAPK1 115 38 15 MAPK2 108 9048 MAPK2 99 78 45 MAPKAP-K2 67 12 1 MAPKAP-K3 82 28 1 MARK1 52 20 4 MEK1117 94 41 MELK 61 27 2 Mer 95 74 5 Met 168 21 7 MINK 79 57 18 MKK4 103135 13 MKK6 113 105 50 MKK7β 91 44 9 MLCK 83 38 52 MLK1 92 75 42 Mnk2103 71 29 MRCKβ 95 52 18 MRCKα 96 76 32 MSK1 105 97 33 MSK2 56 22 12MSSK1 12 4 4 MST1 58 36 17 MST2 106 104 38 MST3 50 10 2 MuSK 97 83 63NEK11 89 58 19 NEK2 99 100 37 NEK3 79 41 18 NEK6 78 43 4 NEK7 110 94 27NLK 103 90 44 p70S6K 43 17 10 PAK2 103 79 16 PAK3 43 5 3 PAK4 99 91 58PAK5 69 6 2 PAK6 77 22 1 PAR-1Bα 70 20 8 PASK 136 114 26 PDGFRβ 59 19 9PDGFRα(D842V) 60 11 5 PDGFRα 100 106 51 PDGFRα(V561D) 59 11 7 PDK1 97 5716 PhKγ2 67 62 16 Pim-1 44 9 2 Pim-2 82 17 10 PKA(b) 104 52 7 PKA 99 8516 PKBβ 61 9 −1 PKBα 98 67 8 PKBγ 86 50 5 PKCμ 90 81 44 PKCβI 108 112100 PKCβII 71 47 30 PKCα 75 34 32 PKCγ 72 47 27 PKCδ 105 94 63 PKCε 10890 59 PKCζ 34 10 2 PKCη 107 99 84 PKCθ 88 31 21 PKCι 66 69 63 PKD2 106108 81 PKG1β 31 16 5 PKG1α 41 18 7 Plk3 114 106 115 PRAK 18 18 35 PRK292 35 8 PrKX 49 14 16 PTK5 99 95 88 Pyk2 90 45 9 Ret 23 −1 −2 RIPK2 10395 64 ROCK-I 95 90 54 ROCK-II 100 66 39 ROCK-II 91 59 39 Ron 32 2 4 Ros95 40 35 Rse 35 14 0 Rsk1 45 9 4 Rsk1 75 8 5 Rsk2 60 4 3 Rsk3 78 31 7Rsk4 71 25 12 SAPK2a 99 106 106 SAPK2a(T106M) 110 106 80 SAPK2b 99 10077 SAPK3 108 79 40 SAPK4 103 86 57 SGK 89 34 2 SGK2 102 36 5 SGK3 103 9634 SIK 115 28 5 Snk 93 96 61 SRPK1 56 14 6 SRPK2 37 15 4 STK33 100 94 64Syk 2 2 3 TAK1 105 101 86 TAO2 97 64 25 TBK1 37 5 12 Tie2 97 67 7 TrkA20 4 2 TrkB 22 0 0 TSSK1 89 10 5 TSSK2 97 29 2 VRK2 98 88 67 WNK2 96 7521 WNK3 110 98 38 Yes 63 33 3 ZAP-70 57 19 10 ZIPK 104 81 28

TABLE 5 Dose response effect (as % of Control) of IAA on selectedprotein kinases Kinase 1 ug/ml 5 ug/ml 25 ug/ml 50 ug/ml Abl(T315I) 104119 84 56 ALK4 92 110 113 AMPK 122 121 86 49 Aurora-A 103 106 61 20 Bmx90 125 108 43 BTK 96 102 62 48 CaMKI 126 139 146 54 CDK1/cyclinB 96 10286 69 CDK2/cyclinA 102 111 98 59 CDK2/cyclinE 81 89 72 55 CDK3/cyclinE99 121 107 62 CDK5/p25 88 108 95 69 CDK5/p35 92 117 100 73 CDK6/cyclinD3111 119 108 64 CDK9/cyclin T1 87 109 77 51 CHK1 105 117 140 159 CHK2 102106 75 46 CK1(y) 94 105 103 CK1γ1 98 102 69 21 CK1γ2 89 88 39 42 CK1γ391 87 26 17 CK1δ 95 111 90 56 cKit(D816H) 98 117 100 59 CSK 95 111 72 86cSRC 99 111 100 53 DAPK1 73 52 36 21 DAPK2 59 54 50 47 DRAK1 102 123 12975 EphA2 104 118 108 88 EphA8 113 120 117 98 EphB1 112 151 220 208 ErbB493 107 110 20 Fer 95 76 49 38 Fes 101 110 120 59 FGFR2 85 122 97 5 Fgr99 120 119 70 Flt4 85 37 74 33 Fyn 90 88 92 90 GSK3β 86 77 47 14 GSK3α85 83 56 17 Hck 88 81 76 4 HIPK2 101 107 107 84 HIPK3 97 101 127 84IGF-1R 132 229 278 301 IKKβ 103 116 93 56 IR 110 107 121 131 IRAK1 115143 156 122 JAK3 88 98 83 74 Lyn 82 114 41 73 MAPK1 81 87 55 55MAPKAP-K2 100 98 82 36 MAPKAP-K3 108 113 106 80 MINK 102 122 118 127MSK1 99 103 66 61 MSK2 95 90 44 45 MSSK1 90 78 52 52 p70S6K 94 98 84 58PAK3 91 66 21 11 PAK5 101 108 106 59 PAK6 98 109 106 102 PhKγ2 103 109102 66 Pim-1 104 106 77 46 Pim-2 101 108 88 60 PKA(b) 104 115 86 12 PKA110 102 99 106 PKBβ 104 110 57 76 PKBα 98 103 91 72 PKBγ 103 108 104 76PKCβII 103 103 102 59 PKCα 106 104 89 46 PRAK 99 91 38 18 PrKX 94 92 9158 Ron 117 113 113 40 Ros 101 108 84 75 Rsk1 96 101 72 48 Rsk2 95 101 7636 SGK 102 110 100 96 SGK2 99 128 105 60 Syk 85 92 53 7 TBK1 100 105 8286 Tie2 101 124 113 40 TrkA 112 139 24 20 TrkB 97 111 90 59 TSSK2 99 112109 75 ZIPK 102 102 95 73

TABLE 6 Dose response effect (as % of Control) of HHIAA on selectedprotein kinases Kinase 1 ug/ml 5 ug/ml 25 ug/ml 50 ug/ml Abl(T315I) 113109 84 38 ALK4 123 121 108 AMPK 133 130 137 87 Aurora-A 111 107 64 27Bmx 103 102 106 44 BTK 110 105 67 61 CaMKI 148 151 140 56 CDK1/cyclinB118 115 98 85 CDK2/cyclinA 109 112 82 60 CDK2/cyclinE 83 84 70 88CDK3/cyclinE 115 119 108 85 CDK5/p25 101 94 69 51 CDK5/p35 110 103 73 68CDK6/cyclinD3 119 124 117 83 CDK9/cyclin T1 106 96 66 40 CHK1 127 124140 144 CHK2 119 117 110 82 CK1(y) 102 102 100 CK1γ1 105 103 68 30 CK1γ299 99 45 49 CK1γ3 104 98 28 22 CK1δ 110 115 89 56 cKit(D816H) 116 109 9168 CSK 100 108 109 112 cSRC 105 114 103 37 DAPK1 94 67 37 27 DAPK2 72 5846 47 DRAK1 110 119 103 69 EphA2 106 127 115 68 EphA8 133 109 89 74EphB1 154 162 200 164 ErbB4 141 122 85 14 Fer 90 62 13 20 Fes 137 126111 81 FGFR2 116 120 71 7 Fgr 122 127 118 91 Flt4 135 116 88 58 Fyn 104119 82 81 GSK3β 138 84 51 10 GSK3α 89 82 58 18 Hck 93 99 73 77 HIPK2 103105 100 98 HIPK3 117 121 118 29 IGF-1R 138 173 207 159 IKKβ 123 116 9879 IR 129 95 105 81 IRAK1 142 140 152 120 JAK3 104 103 61 90 Lyn 115 11356 80 MAPK1 100 88 55 67 MAPKAP-K2 104 99 71 29 MAPKAP-K3 111 109 99 77MINK 107 102 114 123 MSK1 105 101 58 69 MSK2 101 86 39 48 MSSK1 98 78 4160 p70S6K 108 99 78 56 PAK3 113 24 14 10 PAK5 109 105 89 36 PAK6 106 10688 71 PhKγ2 105 109 85 54 Pim-1 107 110 81 50 Pim-2 111 106 98 58 PKA(b)105 119 67 12 PKA 98 107 102 91 PKBβ 121 142 50 42 PKBα 105 108 81 57PKBγ 115 116 107 42 PKCβII 113 115 109 95 PKCα 110 90 105 103 PRAK 10989 41 33 PrKX 86 88 77 59 Ron 114 106 129 74 Ros 113 107 109 98 Rsk1 101102 53 60 Rsk2 105 103 58 25 SGK 108 114 112 64 SGK2 120 121 96 63 Syk100 95 68 17 TBK1 115 103 99 114 Tie2 109 120 95 43 TrkA 87 73 41 24TrkB 100 107 97 13 TSSK2 115 112 109 71 ZIPK 109 109 96 8

TABLE 7 Dose response effect (as % of Control) of beta acids on selectedprotein kinases Kinase 1 ug/ml 5 ug/ml 25 ug/ml 50 ug/ml Abl(T315I) 101101 70 29 ALK4 108 114 90 AMPK 136 131 135 77 Aurora-A 110 85 43 2 Bmx111 100 93 54 BTK 96 90 14 37 CaMKI 142 142 131 57 CDK1/cyclinB 116 12095 65 CDK2/cyclinA 106 104 94 64 CDK2/cyclinE 93 86 81 65 CDK3/cyclinE119 115 96 53 CDK5/p25 97 97 95 96 CDK5/p35 109 106 90 50 CDK6/cyclinD3107 117 101 76 CDK9/cyclin T1 101 104 88 35 CHK1 111 125 144 164 CHK2103 100 94 69 CK1(y) 102 104 83 CK1γ1 100 95 82 33 CK1γ2 97 83 55 44CK1γ3 99 75 40 21 CK1δ 103 98 81 54 cKit(D816H) 103 112 100 18 CSK 107111 108 145 cSRC 104 99 90 19 DAPK1 109 106 88 59 DAPK2 97 76 57 45DRAK1 124 134 107 51 EphA2 116 122 115 80 EphA8 107 105 86 36 EphB1 130164 204 207 ErbB4 111 118 116 28 Fer 78 69 30 18 Fes 120 106 114 79FGFR2 130 118 99 7 Fgr 119 119 127 62 Flt4 104 96 65 22 Fyn 99 94 86 78GSK3β 83 67 27 4 GSK3α 70 71 31 1 Hck 102 88 61 22 HIPK2 101 104 99 94HIPK3 109 119 118 83 IGF-1R 101 163 262 260 IKKβ 110 113 85 59 IR 106106 108 95 IRAK1 143 155 165 158 JAK3 100 98 64 38 Lyn 114 120 68 59MAPK1 88 75 51 37 MAPKAP-K2 111 104 65 22 MAPKAP-K3 108 106 102 69 MINK102 103 123 140 MSK1 106 97 54 36 MSK2 96 86 28 25 MSSK1 95 82 61 67p70S6K 89 95 69 44 PAK3 103 40 16 11 PAK5 103 99 81 44 PAK6 103 98 82 83PhKγ2 108 103 79 40 Pim-1 104 97 57 21 Pim-2 103 101 68 73 PKA(b) 120104 51 3 PKA 103 105 102 28 PKBβ 114 108 56 52 PKBα 98 95 80 58 PKBγ 105104 101 52 PKCβII 107 105 100 49 PKCα 108 104 98 54 PRAK 105 81 24 11PrKX 93 86 68 29 Ron 108 119 98 44 Ros 107 103 80 98 Rsk1 103 99 69 17Rsk2 98 96 56 8 SGK 109 111 98 100 SGK2 123 113 84 0 Syk 92 81 62 16TBK1 110 103 80 78 Tie2 110 100 106 79 TrkA 97 66 53 18 TrkB 105 100 8611 TSSK2 112 109 103 62 ZIPK 105 110 85 37

TABLE 8 Dose response effect (as % of Control) of xanthohumol onselected protein kinases Kinase 1 ug/ml 5 ug/ml 25 ug/ml 50 ug/mlAbl(T315I) 126 115 16 4 ALK4 116 100 71 49 AMPK 122 113 90 81 Aurora-A83 27 3 8 Bmx 108 97 22 0 BTK 109 57 2 20 CaMKI 142 83 3 4 CDK1/cyclinB118 103 46 18 CDK2/cyclinA 107 96 57 6 CDK2/cyclinE 82 86 18 9CDK3/cyclinE 101 100 37 8 CDK5/p25 97 97 24 87 CDK5/p35 103 102 41 44CDK6/cyclinD3 110 79 23 7 CDK9/cyclin T1 110 107 45 31 CHK1 121 126 142149 CHK2 25 5 3 2 CK1(y) 91 63 37 9 CK1γ1 101 79 50 26 CK1γ2 92 48 30 12CK1γ3 98 51 22 15 CK1δ 75 32 16 12 cKit(D816H) 94 45 14 CSK 113 113 93100 cSRC 92 50 27 21 DAPK1 113 85 49 20 DAPK2 105 88 45 26 DRAK1 133 4019 −5 EphA2 124 113 121 52 EphA8 103 92 29 19 EphB1 92 122 175 161 ErbB4132 85 52 27 Fer 55 20 10 1 Fes 131 106 102 26 FGFR2 116 89 36 4 Fgr 10136 10 0 Flt4 74 10 11 4 Fyn 104 66 42 18 GSK3β 120 99 25 3 GSK3α 102 8111 −4 Hck 85 35 17 0 HIPK2 110 98 75 37 HIPK3 106 102 90 59 IGF-1R 107113 129 139 IKKβ 145 118 61 44 IR 120 108 97 103 IRAK1 129 104 81 36JAK3 104 84 17 5 Lyn 97 40 4 2 MAPK1 91 64 19 17 MAPKAP-K2 99 95 6 8MAPKAP-K3 100 99 17 7 MINK 42 10 5 7 MSK1 114 92 31 9 MSK2 126 61 8 19MSSK1 47 11 7 5 p70S6K 94 48 19 7 PAK3 21 18 8 4 PAK5 106 99 42 5 PAK6105 94 14 2 PhKγ2 106 60 11 5 Pim-1 88 35 4 3 Pim-2 104 48 14 6 PKA(b)137 113 33 2 PKA 105 109 98 21 PKBβ 146 102 1 8 PKBα 102 81 18 5 PKBγ104 104 12 4 PKCβII 108 108 71 79 PKCα 100 100 75 83 PRAK 101 53 2 2PrKX 92 75 2 3 Ron 135 127 60 69 Ros 101 99 85 94 Rsk1 34 49 4 0 Rsk2 9643 3 4 SGK 111 84 0 3 SGK2 130 110 2 −4 Syk 95 60 32 17 TBK1 104 71 4542 Tie2 94 96 100 35 TrkA 36 19 8 3 TrkB 95 89 58 3 TSSK2 102 95 61 48ZIPK 115 74 20 70

Results—The effect on kinase activity modulation by the variouscompounds tested displayed a wide range of modulatory effects dependingon the specific kinase and compound tested (Tables 2-8) withrepresentative examples enumerated below.

PI3Kδ, a kinase strongly implicated in autoimmune diseases such as, forexample, rheumatoid arthritis and lupus erythematosus, exhibited aresponse inhibiting 36%, 78% and 87% of kinase activity at 10, 50, and100 ug/mil respectively for MgRho. MgRho inhibited Syk in a dosedependent manner with 21%, 54% and 72% inhibition at 10, 50, and 100μg/ml respectively. Additionally, GSK or glycogen synthase kinase (bothGSK alpha and beta) displayed inhibition following mgRho exposure(alpha, 35, 36, 87% inhibition; beta, 35, 83, 74% inhibitionrespectively at 10, 50, 100 μg/ml). See Table 2.

THIAA displayed a dose dependent inhibition of kinase activity for manyof the kinases examined with inhibition of FGFR2 of 7%, 16%, 77%, and91% at 1, 5, 25, and 50 μg/ml respectively. Similar results wereobserved for FGFR3 (0%, 6%, 61%, and 84%) and TrkA (24%, 45%, 93%, and94%) at 1, 5, 25, and 50 μg/ml respectively. See Table 3.

The acacia extract tested (A. nilotica) appeared to be the most potentinhibitor of kinase activity examined (Table 4), demonstrating 80% orgreater inhibition of activity for such kinases as Syk (98%), Lyn (96%),GSK3a (95%), Aurora-A (92%), Flt4 (88%), MSSK1 (88%), GSK3B (87%), BTK(85%), PRAK (82%), and TrkA (80%), all at a 1 μg/ml exposure.

Example 3 Effect of Hops Components on PI3K Activity

The inhibitory effect on human PI3K-β, PI3K-γ, and PI3K-δ of the hopscomponents xanthohumol and the magnesium salts of beta acids, isoalphaacids (Mg-IAA), tetrahydro-isoalpha acids (Mg-THIAA), andhexahydro-isoalpha acids (Mg-HHIAA) were examined according to theprocedures and protocols of Example 1. Additionally examined was anAcacia nilotica heartwood extract. All compounds were tested at 50μg/ml. The results are presented graphically as FIG. 3. It should benoted that all of the hops compounds tested showed >50% inhibition ofPI3K activity with Mg-THIAA producing the greatest overall inhibition(>80% inhibition for all PI3K isoforms tested). Further note that bothxanthohumol and Mg-beta acids were more inhibitory to PI3K-γ than toPI3K-β or PI3K-δ. Mg-IAA was approximately 3-fold more inhibitory toPI3K-β than to PI3K-γ or PI3K-δ. The Acacia nilotica heartwood extractappeared to stimulate PI3K-β or PI3K-δ activity. Comparable results wereobtained for Syk and GSK kinases (data not shown).

Example 4 Inhibition of PGE₂ Synthesis in Stimulated and NonstimmulatedMurine Macrophages by Hops Compounds and Derivatives

The objective of this example was to assess the extent to which hopsderivatives inhibited COX-2 synthesis of PGE₂ preferentially over COX-1synthesis of PGE₂ in the murine RAW 264.7 macrophage model. The RAW264.7 cell line is a well-established model for assessinganti-inflammatory activity of test agents. Stimulation of RAW 264.7cells with bacterial lipopolysaccharide induces the expression of COX-2and production of PGE₂. Inhibition of PGE₂ synthesis is used as a metricfor anti-inflammatory activity of the test agent. Equipment, Chemicalsand Reagents, PGE₂ assay, and calculations are described below.

Equipment—Equipment used in this example included an OHAS Model #E01140analytical balance, a Form a Model #F1214 biosafety cabinet (Marietta,Ohio), various pipettes to deliver 0.1 to 100 μl (VWR, Rochester, N.Y.),a cell hand tally counter (VWR Catalog #23609-102, Rochester, N.Y.), aForm a Model #F3210 CO₂ incubator (Marietta, Ohio), a hemocytometer(Hausser Model #1492, Horsham, Pa.), a Leica Model #DM IL invertedmicroscope (Wetzlar, Germany), a PURELAB Plus Water Polishing System(U.S. Filter, Lowell, Mass.), a 4° C. refrigerator (Form a Model #F3775,Marietta, Ohio), a vortex mixer (VWR Catalog #33994-306, Rochester,N.Y.), and a 37° C. water bath (Shel Lab Model #1203, Cornelius, Oreg.).

Chemicals and Reagents—Bacterial lipopolysaccharide (LPS; B E. coli055:B5) was from Sigma (St. Louis, Mo.). Heat inactivated Fetal BovineSerum (FBS-HI Cat. #35-011CV), and Dulbecco's Modification of Eagle'sMedium (DMEM Cat #10-013CV) was purchased from Mediatech (Herndon, Va.).Hops fractions (1) alpha hop (1% alpha acids; AA), (2) aromahop OE (10%beta acids and 2% isomerized alpha acids, (3) isohop (isomerized alphaacids; IAA), (4) beta acid solution (beta acids BA), (5) hexahop gold(hexahydro isomerized alpha acids; HHIAA), (6) redihop (reducedisomerized-alpha acids; RIAA), (7) tetrahop (tetrahydro-iso-alpha acidsTHIAA) and (8) spent hops were obtained from Betatech Hops Products(Washington, D.C., U.S.A.). The spent hops were extracted two times withequal volumes of absolute ethanol. The ethanol was removed by heating at40° C. until a only thick brown residue remained. This residue wasdissolved in DMSO for testing in RAW 264.7 cells.

Test materials—Hops derivatives as described in Table 12 were used. TheCOX-1 selective inhibitor aspirin and COX-2 selective inhibitorcelecoxib were used as positive controls. Aspirin was obtained fromSigma (St. Louis, Mo.) and the commercial formulation of celecoxib wasused (Celebrex™, Searle & Co., Chicago, Ill.).

Cell culture and treatment with test material—RAW 264.7 cells, obtainedfrom American Type Culture Collection (Catalog #TIB-71, Manassas, Va.),were grown in Dulbecco's Modification of Eagle's Medium (DMEM,Mediatech, Herndon, Va.) and maintained in log phase. The DMEM growthmedium was made by adding 50 ml of heat inactivated FBS and 5 ml ofpenicillin/streptomycin to a 500 ml bottle of DMEM and storing at 4° C.The growth medium was warmed to 37° C. in water bath before use.

For COX-2 associated PGE₂ synthesis, 100 μl of medium was removed fromeach well of the cell plates prepared on day one and replaced with 100μl of equilibrated 2× final concentration of the test compounds. Cellswere then incubated for 90 minutes. Twenty μl of LPS were added to eachwell of cells to be stimulated to achieve a final concentration of 1 μgLPS/ml and the cells were incubated for 4 h. The cells were furtherincubated with 5 μM arachadonic acid for 15 minutes. Twenty-five μl ofsupernatant medium from each well was transferred to a clean microfugetube for the determination of PGE₂ released into the medium.

For COX-1 associated PGE₂ synthesis, 100 μl of medium were removed fromeach well of the cell plates prepared on day one and replaced with 100μl of equilibrated 2× final concentration of the test compounds. Cellswere then incubated for 90 minutes. Next, instead of LPS stimulation,the cells were incubated with 100 μM arachadonic acid for 15 minutes.Twenty-five μl of supernatant medium from each well was transferred to aclean microfuge tube for the determination of PGE₂ released into themedium.

The appearance of the cells was observed and viability was assessedvisually. No apparent toxicity was observed at the highestconcentrations tested for any of the compounds. Twenty-five μl ofsupernatant medium from each well was transferred to a clean microfugetube for the determination of PGE₂ released into the medium. PGE₂ wasdetermined and reported as previously described below.

PGE₂ assay—A commercial, non-radioactive procedure for quantification ofPGE₂ was employed (Caymen Chemical, Ann Arbor, Mich.) and therecommended procedure of the manufacturer was used without modification.Briefly, 25 μl of the medium, along with a serial dilution of PGE₂standard samples, were mixed with appropriate amounts ofacetylcholinesterase-labeled tracer and PGE₂ antiserum, and incubated atroom temperature for 18 h. After the wells were emptied and rinsed withwash buffer, 200 μl of Ellman's reagent containing substrate foracetylcholinesterase were added. The reaction was maintained on a slowshaker at room temperature for 1 h and the absorbance at 415 nm wasdetermined in a Bio-Tek Instruments (Model #Elx800, Winooski, Vt.) ELISAplate reader. The PGE₂ concentration was represented as picograms perml. The manufacturer's specifications for this assay include anintra-assay coefficient of variation of <10%, cross reactivity with PGD₂and PGF₂ of less than 1% and linearity over the range of 10-1000 pgml⁻1. The median inhibitory concentrations (IC₅₀) for PGE₂ synthesisfrom both COX-2 and COX-1 were calculated as described below.

Calculations—The median inhibitory concentrations (IC₅₀) for PGE₂synthesis were calculated using CalcuSyn (BIOSOFT, Ferguson, Mo.). Aminimum of four concentrations of each test material or positive controlwas used for computation. This statistical package performs multipledrug dose-effect calculations using the Median Effect methods describedby T. C Chou and P. Talalay [Chou, T. C. and P. Talalay. Quantitativeanalysis of dose-effect relationships; the combined effects of multipledrugs or enzyme inhibitors. Adv Enzyme Regul 22: 27-55, (1984)] and isincorporated herein by reference. Experiments were repeated three timeson three different dates. The percent inhibition at each dose wasaveraged over the three independent experiments and used to calculatethe median inhibitory concentrations reported.

Median inhibitory concentrations were ranked into four arbitrarycategories: (1) highest anti-inflammatory response for those agents withan IC₅₀ values within 0.3 μg/ml of 0.1; (2) high anti-inflammatoryresponse for those agents with an IC₅₀ value within 0.7 μg/ml of 1.0;(3) intermediate anti-inflammatory response for those agents with IC₅₀values between 2 and 7 μg/ml; and (4) low anti-inflammatory response forthose agents with IC₅₀ values greater than 12 μg/ml, the highestconcentration tested

Results—The aspirin and celecoxib positive controls demonstrated theirrespective cyclooxygenase selectivity in this model system (Table 9).While aspirin was approximately 1000-fold more selective for COX-1,celecoxib was 114 times more selective for COX-2. All hops materialswere COX-2 selective with Rho isoalpha acids and isoalpha acidsdemonstrating the highest COX-2 selectivity, 363- and 138-foldrespectively. Such high COX-2 selectivity combined with low medianinhibitory concentrations, has not been previously reported for naturalproducts from other sources. Of the remaining hops derivatives, only thearomahop oil exhibited a marginal COX-2 selectivity of 3-fold. Forextrapolating in vitro data to clinical efficacy, it is generallyassumed that a COX-2 selectivity of 5-fold or greater indicates thepotential for clinically significant protection of gastric mucosa. Underthis criterion, beta acids, CO₂ hop extract, spent hops CO₂/ethanol,tetrahydro isoalpha acids and hexahydro isoalpha acids displayedpotentially clinically relevant COX-2 selectivity.

TABLE 9 COX-2 and COX-1 inhibition in RAW 264.7 cells by hop fractionsand derivatives IC₅₀ COX-2 IC₅₀ COX-1 [μg/ml] [μg/ml] COX-1/COX-2 TestMaterial Rho Isoalpha acids 0.08 29 363 Isoalpha acids 0.13 18 138 Betaacids 0.54 29 54 CO₂ hop extract 0.22 6.3 29 Alpha acids 0.26 6.2 24Spent hops CO₂/Ethanol 0.88 21 24 Tetrahydro isoalpha acids 0.20 4.0 20Hexahydro isoalpha acids 0.29 3.0 10 Aromahop Oil 1.6 4.1 3.0 PositiveControls Aspirin 1.16 0.0009 0.0008 Celecoxib 0.005 0.57 114

Example 5 Lack of Direct PGE₂ Inhibition by Reduced Isomerized AlphaAcids or Isomerized Alpha Acids in LPS-Stimulated Raw 264.7 Cells

The objective of this study was to assess the ability of the hopsderivatives reduced isoalpha acids and isomerized alpha acids tofunction independently as direct inhibitors of COX-2 mediated PGE₂biosynthesis in the RAW 264.7 cell model of inflammation. The RAW 264.7cell line as described in Example 4 was used in this example. Equipment,chemicals and reagents, PGE₂ assay, and calculations were as describedin Example 4.

Test Materials—Hops derivatives reduced isoalpha acids and isomerizedalpha acids, as described in Table 12, were used. Aspirin, a COX-1selective positive control, was obtained from Sigma (St. Louis, Mo.).

Cell culture and treatment with test material —RAW 264.7 cells (TE3-71)were obtained from the American Type Culture Collection (Manassas, Va.)and sub-cultured as described in Example 4. Following overnightincubation at 37° C. with 5% CO₂, the growth medium was aspirated andreplaced with 200 μl DMEM without FBS or penicillin/streptomycin. RAW264.7 cells were stimulated with LPS and incubated overnight to induceCOX-2 expression. Eighteen hours post LPS-stimulation, test materialswere added followed 60 minutes later by the addition of the calciumionophore A23187. Test materials were dissolved in DMSO as a 250-foldstock solution. Four μl of this 250-fold stock test material preparationwas added to 1 ml of DMEM and 200 μl of this solution was subsequentlyadded to eight wells for each dose of test material. Supernatant mediawas sampled for PGE₂ determination after 30 minutes. Median inhibitoryconcentrations were computed from a minimum of four concentrations overtwo independent experiments as described in Example 4.

Determination of PGE₂—A commercial, non-radioactive procedure forquantification of PGE₂ was employed (Caymen Chemical, Ann Arbor, Mich.)for the determination of PGE₂ and the recommended procedure of themanufacturer was used without modification as described in Example 4.

Cell viability—Cell viability was assessed by microscopic inspection ofcells prior to or immediately following sampling of the medium for PGE₂assay. No apparent cell mortality was noted at any of the concentrationstested.

Calculations—Four concentrations 0.10, 1.0, 10 and 100 μg/ml were usedto derive dose-response curves and compute medium inhibitoryconcentrations (IC_(50s)) with 95% confidence intervals using CalcuSyn(BIOSOFT, Ferguson, Mo.).

Results—LPS-stimulation of PGE₂ production in RAW 264.7 cells rangedfrom 14-fold to 21-fold relative to non-stimulated cells. The IC₅₀ valueof 8.7 μg/ml (95% CL=3.9−19) computed for the aspirin positive controlwas consistent with published values for direct COX-2 inhibition rangingfrom 1.4 to 50 μg/ml [Warner, T. D. et al. Nonsteroidal drugselectivities for cyclo-oxygenase-1 rather than cyclo-oxygenase-2 areassociated with human gastrointestinal toxicity: A full in vitroanalysis. Proc. Natl. Acad. Sci. USA 96:7563-7568, (1999)] andhistorical data of this laboratory of 3.2 μg/ml (95% CL=0.55−19) in theA549 cell line.

When added following COX-2 induction in RAW 264.7 cells by LPS, bothRIAA and IAA produced only modest, dose-related inhibition of PGE₂. Overthe 1000-fold increase in concentration of test material, only a 14 and10 percent increase in inhibition was noted, respectively, for RIAA andIAA. The shallowness of the dose-response slopes resulted in IC₅₀ values(Table 10) in the mg/ml range for RIAA (36 mg/ml) and IAA (>1000 mg/ml).The minimal changes observed in response over three-log units of dosessuggests that the observed PGE₂ inhibitory effect of the hopsderivatives in this cell-based assay may be a secondary effect on thecells and not a direct inhibition of COX-2 enzyme activity.

FIGS. 4A and 4B depict the dose-response data respectively, for RIAA andIAA as white bars and the dose-response data from this example as graybars. The effect of sequence of addition is clearly seen and supportsthe inference that RIAA and IAA are not direct COX-2 enzyme inhibitors.

It appears that (1) hop materials were among the most active,anti-inflammatory natural products tested as assessed by their abilityto inhibit PGE₂ biosynthesis in vitro; (2) RIAA and IAA do not appear tobe direct COX-2 enzyme inhibitors based on their pattern of inhibitionwith respect to COX-2 induction; and (3) RIAA and IAA have a COX-2selectively that appears to be based on inhibition of COX-2 expression,not COX-2 enzyme inhibition. This selectivity differs from celecoxib,whose selectivity is based on differential enzyme inhibition.

TABLE 10 Median inhibitory concentrations for RIAA, IAA in RAW 264.7cells when test material is added post overnight LPS-stimulation. IC₅₀95% Confidence Interval [μg/ml] [μg/ml] Test Material RIAA 36,00017,000-79,000 IAA >1,000,000 — Positive Control Aspirin 8.7 μg/ml3.9-19  RAW 264.7 cells were stimulated with LPS and incubated overnightto induce COX-2 expression. Eighteen hours post LPS-stimulation, testmaterial was added followed 60 minutes later by the addition of A23187.Supernatant media was sampled for PGE₂ determination after 30 minutes.Median inhibitory concentrations were computed from a minimum of eightreplicates at four concentrations over two independent experiments.

Example 6 Hops Compounds and Derivatives are not Direct CyclooxygenaseEnzyme Inhibitors in A549 Pulmonary Epithelial Cells

Chemicals—Hops and hops derivatives used in this example were previouslydescribed in Example 4. All other chemicals were obtained from suppliersas described in Example 4.

Equipment, PGE₂ assay, and Calculations were as described in Example 4.

Cells—A549 (human pulmonary epithelial) cells were obtained from theAmerican Type Culture Collection (Manassas, Va.) and sub-culturedaccording to the instructions of the supplier. The cells were routinelycultured at 37° C. with 5% CO₂ in RPMI 1640 containing 10% FBS, with 50units penicillin/ml, 50 μg streptomycin/ml, 5 mM sodium pyruvate, and 5mM L-glutamine. On the day of the experiments, exponentially growingcells were harvested and washed with serum-free RPMI 1640.

Log phase A549 cells were plated at 8×10⁴ cells per well in 0.2 mlgrowth medium per well in a 96-well tissue culture plate. For thedetermination of PGE₂ inhibition by the test compounds, the procedure ofWarner, et al. [Nonsteroid drug selectivities for cyclo-oxygenase-1rather than cyclo-oxygenase-2 are associated with human gastrointestinaltoxicity: a full in vitro analysis. Proc Natl Acad Sci USA 96,7563-7568, (1999)], also known as the WHMA-COX-2 protocol was followedwith no modification. Briefly, 24 hours after plating of the A549 cells,interleukin-1β (10 ng/ml) was added to induce the expression of COX-2.After 24 hr, the cells were washed with serum-free RPMI 1640.Subsequently, the test materials, dissolved in DMSO and serum-free RPMI,were added to the wells to achieve final concentrations of 25, 5.0, 0.5and 0.05 μg/ml. Each concentration was run in duplicate. DMSO was addedto the control wells in an equal volume to that contained in the testwells. Sixty minutes later, A23187 (50 μM) was added to the wells torelease arachadonic acid. Twenty-five μl of media were sampled from thewells 30 minutes later for PGE₂ determination.

Cell viability was assessed visually and no apparent toxicity wasobserved at the highest concentrations tested for any of the compounds.PGE₂ in the supernatant medium was determined and reported as previouslydescribed in Example 4. The median inhibitory concentration (IC₅₀) forPGE₂ synthesis was calculated as previously described in Example 4.

Results—At the doses tested, the experimental protocol failed to capturea median effective concentration for any of the hops extracts orderivatives. Since the protocol requires the stimulation of COX-2expression prior to the addition of the test compounds, it is believedthat the failure of the test materials to inhibit PGE₂ synthesis is thattheir mechanism of action is to inhibit the expression of the COX-2isozyme and not activity directly. While some direct inhibition wasobserved using the WHMA-COX-2 protocol, this procedure appearsinappropriate in evaluating the anti-inflammatory properties of hopscompounds or derivatives of hops compounds.

Example 7 Hops Derivatives Inhibit Mite Dust Allergen Activation of PGE₂Biosynthesis in A549 Pulmonary Epithelial Cells

Chemicals—Hops and hops derivatives, (1) alpha hop (1% alpha acids; AA),(2) aromahop OE (10% beta acids and 2% isomerized alpha acids, (3)isohop (isomerized alpha acids; IAA), (4) beta acid solution (beta acidsBA), (5) hexahop gold (hexahydro isomerized alpha acids; HHIAA), (6)redihop (reduced isomerized-alpha acids; RIAA), and (7) tetrahop(tetrahydro-iso-alpha acids THIAA), used in this example were previouslydescribed in Example 1. All other chemicals were obtained from suppliersas described in Example 4. Test materials at a final concentration of 10μg/ml were added 60 minutes prior to the addition of the mite dustallergen.

Equipment, PGE₂ assay, and Calculations were as described in Example 4.

Mite dust allergen isolation—Dermatophagoides farinae is the Americanhouse dust mite. D. farinae were raised on a 1:1 ratio of PurinaLaboratory Chow (Ralston Purina, Co, St. Louis, Mo.) and Fleischmann'sgranulated dry yeast (Standard Brands, Inc. New York, N.Y.) at roomtemperature and 75% humidity. Live mites were aspirated from the culturecontainer as they migrated from the medium, killed by freezing,desiccated and stored at 0% humidity. The allergenic component of themite dust was extracted with water at ambient temperature. Five-hundredmg of mite powder were added to 5 ml of water (1:10 w/v) in a 15 mlconical centrifuge tube (VWR, Rochester, N.Y.), shaken for one minuteand allowed to stand overnight at ambient temperature. The next day, theaqueous phase was filtered using a 0.2 μm disposable syringe filter(Nalgene, Rochester, N.Y.). The filtrate was termed mite dust allergenand used to test for induction of PGE₂ biosynthesis in A549 pulmonaryepithelial cells.

Cell culture and treatment—The human airway epithelial cell line, A549(American Type Culture Collection, Bethesda, Md.) was cultured andtreated as previously described in Example 6. Mite allergen was added tothe culture medium to achieve a final concentration of 1000 ng/ml.Eighteen hours later, the media were sampled for PGE₂ determination.

Results—Table 11 depicts the extent of inhibition by hops derivatives ofPGE₂ biosynthesis in A549 pulmonary cells stimulated by mite dustallergen. All hops derivatives tested were capable of significantlyinhibiting the stimulatory effects of mite dust allergens.

TABLE 11 PGE₂ inhibition by hops derivatives in A549 pulmonaryepithelial cells stimulated by mite dust allergen. Test Material PercentPGE₂ Inhibition Alpha hop (AA) 81 Aromahop OE 84 Isohop (IAA) 78 Betaacids (BA) 83 Hexahop (HHIAA) 82 Redihop (RIAA) 81 Tetrahop (THIAA) 76

This example illustrates that hops derivatives are capable of inhibitingthe PGE₂ stimulatory effects of mite dust allergens in A549 pulmonarycells.

Example 8 Lack of Direct COX-2 Inhibition by Reduced Isoalpha Acids

The objective of this example was to determine whether magnesium reducedisoalpha acids can act as a direct inhibitor of COX-2 enzymaticactivity.

Materials—Test compounds were prepared in dimethyl sulfoxide (DMSO) andstored at −20° C. LPS was purchased from Sigma-Aldrich (St. Louis, Mo.).MgRIAA was supplied by Metagenics (San Clemente, Calif.), and thecommercial formulation of celecoxib was used (Celebrex™, Searle & Co.,Chicago, Ill.).

Cell Culture—The murine macrophage RAW 264.7 cell line was purchasedfrom ATCC (Manassas, Va.) and maintained according to theirinstructions. Cells were subcultured in 96-well plates at a density of8×10⁴ cells per well and allowed to reach 90% confluence, approximately2 days. LPS (1 μg/ml) or PBS alone was added to the cell media andincubated for 12 hrs. The media was removed from the wells and LPS (1μg/ml) with the test compounds dissolved in DMSO and serum-free RPMI,were added to the wells to achieve final concentrations of MgRIAA at 20,5.0, 1.0 and 0.1 μg/ml and celecoxib at 100, 10, 1 and 0.1 ng/ml. Eachconcentration was run in 8 duplicates. Following 1 hr of incubation withthe test compounds, the cell media were removed and replaced with freshmedia with test compounds with LPS (1 μg/ml) and incubated for 1 hr. Themedia were removed from the wells and analyzed for the PGE₂ synthesis.

PGE₂ assay—A commercial, non-radioactive procedure for quantification ofPGE₂ was employed (Cayman Chemical, Ann Arbor, Mich.). Samples werediluted 10 times in EIA buffer and the recommended procedure of themanufacturer was used without modification. The PGE₂ concentration wasrepresented as picograms per ml. The manufacturer's specifications forthis assay include an intra-assay coefficient of variation of <10%,cross reactivity with PGD₂ and PGF₂ of less than 1% and linearity overthe range of 10-1000 pg ml⁻¹.

COX-2 specific inhibitor celecoxib dose-dependently inhibited COX-2mediated PGE₂ synthesis (100, 10, 1 and 0.1 ng/ml) while no significantPGE₂ inhibition was observed with MgRIAA. The data suggest that MgRIAAis not a direct COX-2 enzymatic inhibitor like celocoxib (FIG. 5)

Example 9 Inhibition of iNOS and COX-2 Protein Expression by MgRIAA

Cellular extracts from RAW 264.7 cells treated with MgRIAA andstimulated with LPS were assayed for iNOS and COX-2 protein by Westernblot.

Materials—Test compounds were prepared in dimethyl sulfoxide (DMSO) andstored at −20° C. MgRIAA was supplied by Metagenics (San Clemente,Calif.). Parthenolide was purchased from Sigma-Aldrich (St. Louis, Mo.).The PI3K inhibitors wortmannin and LY294002 were purchased from EMDBiosciences (San Diego, Calif.). Antibodies generated against COX-2 andiNOS were purchased from Cayman Chemical (Ann Arbor, Mich.). Antibodiesgenerated against GAPDH were purchased from Novus Biological (Littleton,Colo.). Secondary antibodies coupled to horseradish peroxidase werepurchased from Amersham Biosciences (Piscataway, N.J.).

Cell Culture—The murine macrophage RAW 264.7 cell line was purchasedfrom ATCC (Manassas, Va.) and maintained according to theirinstructions. Cells were grown and subcultured in 24-well plates at adensity of 3×10⁵ cells per well and allowed to reach 90% confluence,approximately 2 days. Test compounds were added to the cells in serumfree medium at a final concentration of 0.4% DMSO. Following 1 hr ofincubation with the test compounds, LPS (1 μg/ml) or phosphate bufferedsaline alone was added to the cell wells and incubation continued forthe indicated times.

Western Blot—Cell extracts were prepared in Buffer E (50 mM HEPES, pH7.0; 150 mM NaCl; 1% triton X-100; 1 mM sodium orthovanadate; aprotinin5 μg/ml; pepstatin A 1 μg/mil; leupeptin 5 μg/ml; phenylmethanesulfonylfluoride 1 mM). Briefly, cells were washed twice with cold PBS andBuffer E was added. Cells were scraped into a clean tube, following acentrifugation at 14,000 rpm for 10 minutes at 4° C., the supernatantwas taken as total cell extract. Cell extracts (50 μg) wereelectrophoresed through a pre-cast 4%-20% Tris-HCl-Criterion gel(Bio-Rad, Hercules, Calif.) until the front migration dye reached 5 mmfrom the bottom of the gel. The proteins were transferred tonitrocellulose membrane using a semi-dry system from Bio-Rad (Hercules,Calif.). The membrane was washed and blocked with 5% dried milk powderfor 1 hour at room temperature. Incubation with the primary antibodyfollowed by the secondary antibody was each for one hour at roomtemperature. Chemiluminescence was performed using the SuperSignal WestFemto Maximum Sensitivity Substrate from Pierce Biotechnology (Rockford,Ill.) by incubation of equal volume of luminol/enhancer solution andstable peroxide solution for 5 minutes at room temperature. The Westernblot image was captured using a cooled CCD Kodak (Rochester, N.Y.)IS1000 imaging system. Densitometry was performed using Kodak software.

The percent of COX-2 and iNOS protein expression was assessed usingWestern blot detection. The expression of COX-2 was observed after 20hours stimulation with LPS. As compared to the solvent control of DMSO,a reduction of 55% was seen in COX-2 protein expression by MgRIAA (FIG.6). A specific NF-kB inhibitor parthenolide, inhibited proteinexpression 22.5%, while the PI3-kinase inhibitor decreased COX-2expression about 47% (FIG. 6). Additionally, a reduction of 73% of iNOSprotein expression was observed after 20 hr stimulation with LPS (FIG.7) by MgRIAA.

Example 10 NF-κB Nuclear Translocation and DNA Binding

Nuclear extracts from RAW 264.7 cells treated with MgRIAA and stimulatedwith LPS for 4 hours were assayed for NF-κB binding to DNA.

Materials—Test compounds were prepared in dimethyl sulfoxide (DMSO) andstored at −20° C. MgRIAA was supplied by Metagenics (San Clemente,Calif.). Parthenolide, a specific inhibitor for NF-kB activation waspurchased from Sigma-Aldrich (St. Louis, Mo.). The PI3K inhibitorLY294002 was purchased from EMD Biosciences (San Diego, Calif.).

Cell Culture—The murine macrophage RAW 264.7 cell line was purchasedfrom ATCC (Manassas, Va.) and maintained according to theirinstructions. Cells were subcultured in 6-well plates at a density of1.5×10⁶ cells per well and allowed to reach 90% confluence,approximately 2 days. Test compounds MgRIAA (55 and 14 μg/ml),parthenolide (80 μM) and LY294002 (25 μM) were added to the cells inserum free media at a final concentration of 0.4% DMSO. Following 1 hrof incubation with the test compounds, LPS (1 μg/ml) or PBS alone wasadded to the cell media and incubation continued for an additional fourhours.

NF-κB-DNA binding—Nuclear extracts were prepared essentially asdescribed by Dignam, et al [Nucl Acids Res 11:1475-1489, (1983)].Briefly, cells were washed twice with cold PBS, then Buffer A (10 mMHEPES, pH 7.0; 1.5 mM MgCl₂; 10 mM KCl; 0.1% NP-40; aprotinin 5 μg/ml;pepstatin A 1 μg/ml; leupeptin 5 μg/ml; phenylmethanesulfonyl fluoride 1mM) was added and allowed to sit on ice for 15 minutes. Cells were thenscraped into a clean tube and processed through three cycles offreeze/thaw. The supernatant layer following centrifugation at 10,000×gfor 5 min at 4° C. was the cytoplasmic fraction. The remaining pelletwas resuspended in Buffer C (20 mM HEPES, pH 7.0; 1.5 mM KCl; 420 mMKCl; 25% glycerol; 0.2 M EDTA; aprotinin 5 μg/ml; pepstatin A 1 μg/ml;leupeptin 5 μg/ml; phenylmethanesulfonyl fluoride 1 mM) and allowed tosit on ice for 15 minutes. The nuclear extract fraction was collected asthe supernatant layer following centrifugation at 10,000×g for 5 min at4° C. NF-kB DNA binding of the nuclear extracts was assessed using theTransAM NF-κB kit from Active Motif (Carlsbad, Calif.) as permanufacturer's instructions. As seen in FIG. 8, the TransAM kit detectedthe p50 subunit of NF-κB binding to the consensus sequence in a 96-wellformat. Protein concentration was measured (Bio-Rad assay) and 10 μg ofnuclear protein extracts were assayed in duplicate.

Analysis of nuclear extracts (10 μg protein) was performed in duplicateand the results are presented graphically in FIG. 9. Stimulation withLPS (1 μg/ml) resulted in a two-fold increase in NF-κB DNA binding.Treatment with LY294002 (a PI3 kinase inhibitor) resulted in a modestdecrease of NF-κB binding as expected from previous literature reports.Parthenolide also resulted in a significant reduction in NF-κB bindingas expected. A large reduction of NF-κB binding was observed withMgRIAA. The effect was observed in a dose-response manner. The reductionin NF-κB binding may result in reduced transcriptional activation oftarget genes, including COX-2, iNOS and TNFα.

The results suggest that the decreased NF-κB binding observed withMgDHIAA may result in decreased COX-2 protein expression, ultimatelyleading to a decrease in PGE₂ production.

Example 11

Increased Lipogenesis in 3T3-L1 Adipocytes Elicited by a DimethylSulfoxide-Soluble Fraction of an Aqueous Extract of Acacia Bark

The Model—The 3T3-L1 murine fibroblast model is used to study thepotential effects of compounds on adipocyte differentiation andadipogenesis. This cell line allows investigation of stimuli andmechanisms that regulate preadipocytes replication separately from thosethat regulate differentiation to adipocytes [Fasshauer, M., Klein, J.,Neumann, S., Eszlinger, M., and Paschke, R. Hormonal regulation ofadiponectin gene expression in 3T3-L1 adipocytes. Biochem Biophys ResCommun, 290: 1084-1089, (2002); Li, Y. and Lazar, M. A. Differentialgene regulation by PPARgamma agonist and constitutively activePPARgamma2. Mol Endocrinol, 16: 1040-1048, (2002)] as well asinsulin-sensitizing and triglyceride-lowering ability of the test agent[Raz, I., Eldor, R., Cernea, S., and Shafrir, E. Diabetes: insulinresistance and derangements in lipid metabolism. Cure throughintervention in fat transport and storage. Diabetes Metab Res Rev, 21:3-14, (2005)].

As preadipocytes, 3T3-L1 cells have a fibroblastic appearance. Theyreplicate in culture until they form a confluent monolayer, after whichcell-cell contact triggers G₀/G₁ growth arrest. Terminal differentiationof 3T3-L1 cells to adipocytes depends on proliferation of both pre- andpost-confluent preadipocytes. Subsequent stimulation with3-isobutyl-1-methylxanthane, dexamethasone, and high does of insulin(MDI) for two days prompts these cells to undergo post-confluent mitoticclonal expansion, exit the cell cycle, and begin to expressadipocyte-specific genes. Approximately five days after induction ofdifferentiation, more than 90% of the cells display the characteristiclipid-filled adipocyte phenotype. Assessing triglyceride synthesis of3T3-L1 cells provides a validated model of the insulin-sensitizingability of the test agent.

It appears paradoxical that an agent that promotes lipid uptake in fatcells should improve insulin sensitivity. Several hypotheses have beenproposed in an attempt to explain this contradiction. One premise thathas continued to gain research support is the concept of “fatty acidsteal” or the incorporation of fatty acids into the adipocyte from theplasma causing a relative depletion of fatty acids in the muscle with aconcomitant improvement of glucose uptake [Martin, G., K. Schoonjans, etal. PPARgamma activators improve glucose homeostasis by stimulatingfatty acid uptake in the adipocytes. Atherosclerosis 137 Suppl: S75-80,(1998)]. Thiazolidinediones, such as troglitazone and pioglitazone, havebeen shown to selectively stimulate lipogenic activities in fat cellsresulting in greater insulin suppression of lipolysis or release offatty acids into the plasma [Yamauchi, T., J. Kamon, et al. Themechanisms by which both heterozygous peroxisome proliferator-activatedreceptor gamma (PPARgamma) deficiency and PPARgamma agonist improveinsulin resistance. J Biol Chem 276(44): 41245-54, (2001); Oakes, N. D.,P. G. Thalen, et al. Thiazolidinediones increase plasma-adipose tissueFFA exchange capacity and enhance insulin-mediated control of systemicFFA availability. Diabetes 50(5): 1158-65, (2001)]. This action wouldleave less free fatty acids available for other tissues [Yang, W. S., W.J. Lee, et al. Weight reduction increases plasma levels of anadipose-derived anti-inflammatory protein, adiponectin. J ClinEndocrinol Metab 86(8): 3815-9, (2001)]. Thus, insulin desensitizingeffects of free fatty acids in muscle and liver would be reduced as aconsequence of thiazolidinedione treatment. These in vitro results havebeen confirmed clinically [Boden, G. Role of fatty acids in thepathogenesis of insulin resistance and NIDDM. Diabetes 46(1): 3-10,(1997); Stumvoll, M. and H. U. Haring Glitazones: clinical effects andmolecular mechanisms. Ann Med 34(3): 217-24, (2002)].

Test Materials—Troglitazone was obtained from Cayman Chemicals (AnnArbor, Mich., while methylisobutylxanthine, dexamethasone, indomethacin,Oil red 0 and insulin were obtained from Sigma (St. Louis, Mo.). Thetest material was a dark brown powder produced from a 50:50 (v/v)water/alcohol extract of the gum resin of Acacia (AcE) sample #4909 andwas obtained from Bayir Chemicals (No. 68, South Cross Road,Basavanagudi, India). The extract was standardized to contain not lessthan 20% apecatechin. Batch No. A Cat/2304 used in this examplecontained 20.8% apecatechin as determined by UV analysis. Penicillin,streptomycin, Dulbecco's modified Eagle's medium (DMEM) was fromMediatech (Herndon, Va.) and 10% FBS-HI (fetal bovine serum-heatinactivated) from Mediatech and Hyclone (Logan, Utah). All otherstandard reagents, unless otherwise indicted, were purchased from Sigma.

Cell culture and Treatment—The murine fibroblast cell line 3T3-L1 waspurchased from the American Type Culture Collection (Manassas, Va.) andsub-cultured according to instructions from the supplier. Prior toexperiments, cells were cultured in DMEM containing 10% FBS-HI added 50units penicillin/ml and 50 fig streptomycin/ml, and maintained in logphase prior to experimental setup. Cells were grown in a 5% CO₂humidified incubator at 37° C. Components of the pre-confluent mediumincluded (1) 10% FBS/DMEM containing 4.5 g glucose/L; (2) 50 U/milpenicillin; and (3) 50 μg/ml streptomycin. Growth medium was made byadding 50 ml of heat inactivated FBS and 5 ml of penicillin/streptomycinto 500 ml DMEM. This medium was stored at 4° C. Before use, the mediumwas warmed to 37° C. in a water bath.

3T3-T1 cells were seeded at an initial density of 6×10⁴ cells/cm² in24-well plates. For two days, the cells were allowed grow to reachconfluence. Following confluence, the cells were forced to differentiateinto adipocytes by the addition of differentiation medium; this mediumconsisted of (1) 10% FBS/DMEM (high glucose); (2) 0.5 mMmethylisobutylxanthine; (3) 0.5 μM dexamethasone and (4) 10 μg/mlinsulin (MDI medium). After three days, the medium was changed topost-differentiation medium consisting of 10 μg/ml insulin in 10%FBS/DMEM.

AcE was partially dissolved in dimethyl sulfoxide (DMSO) and added tothe culture medium to achieve a concentration of 50 μg/ml at Day 0 ofdifferentiation and throughout the maturation phase (Days 6 or 7(D6/7)). Whenever fresh media were added, fresh test material was alsoadded. DMSO was chosen for its polarity and the fact that it is misciblewith the aqueous cell culture media. As positive controls, indomethacinand troglitazone were added, respectively, to achieve finalconcentrations of 5.0 and 4.4 μg/ml. Differentiated, D6/D7 3T3-L1 cellswere stained with 0.36% Oil Red 0 or 0.001% BODIPY. The completeprocedure for differentiation and treatment of cells with test materialsis outlined schematically in FIG. 10.

Oil Red 0 Staining—Triglyceride content of D6/D7-differentiated 3T3-L1cells was estimated with Oil Red 0 according to the method of Kasturiand Joshi [Kasturi, R. and Joshi, V. C. Hormonal regulation of stearoylcoenzyme A desaturase activity and lipogenesis during adipose conversionof 3T3-L1 cells. J Biol Chem, 257: 12224-12230, 1982]. Monolayer cellswere washed with PBS (phosphate buffered saline, Mediatech) and fixedwith 10% formaldehyde for ten minutes. Fixed cells were stained with anOil Red O working solution of three parts 0.6% Oil Red O/isopropanolstock solution and two parts water for one hour and the excess stain waswashed once with water. The resulting stained oil droplets wereextracted from the cells with isopropanol and quantified byspectrophotometric analysis at 540 nm (MEL312e BIO-KINETICS READER,Bio-Tek Instruments, Winooski, Vt.). Results for test materials and thepositive controls indomethacin and troglitazone were representedrelative to the 540 nm absorbance of the solvent controls.

BODIPYStaining—4,4-Difluoro-1,3,5,7,8-penta-methyl-4-bora-3a,4a-diaza-s-indacene(BODIPY 493/503; Molecular Probes, Eugene, Oreg.) was used forquantification of cellular neutral and nonpolar lipids. Briefly, mediawere removed and cells were washed once with non-sterile PBS. A stock1000× BODIPY/DMSO solution was made by dissolving 1 mg BODIPY in 1 mlDMSO (1,000 μg BODIPY/ml). A working BODIPY solution was then made byadding 10 μl of the stock solution to 990 μl PBS for a final BODIPYconcentration in the working solution of 0.01 μg/μl. One-hundred μl ofthis working solution (1 μg BODIPY) was added to each well of a 96-wellmicrotiter plate. After 15 min on an orbital shaker (DS-500, VWRScientific Products, South Plainfield, N.J.) at ambient temperature, thecells were washed with 100 μl PBS followed by the addition of 100 μl PBSfor reading for spectrofluorometric determination of BODIPYincorporation into the cells. A Packard Fluorocount spectrofluorometer(Model#BF10000, Meridan, Conn.) set at 485 nm excitation and 530 nmemission was used for quantification of BODIPY fluorescence. Results fortest materials, indomethacin, and troglitazone were reported relative tothe fluorescence of the solvent controls.

A chi-square analysis of the relationship between the BODIPYquantification of all neutral and nonpolar lipids and the Oil Red 0determination of triglyceride content in 3T3-L1 cells on D7 indicated asignificant relationship between the two methods with p<0.001 and OddsRatio of 4.64.

Statistical Calculations and Interpretation—AcE and indomethacin wereassayed a minimum of three times in duplicate. Solvent and troglitazonecontrols were replicated eight times also in duplicate. Nonpolar lipidincorporation was represented relative to the nonpolar lipidaccumulation of fully differentiated cells in the solvent controls. Apositive response was defined as an increase in lipid accumulationassessed by Oil Red 0 or BODIPY staining greater than the respectiveupper 95% confidence interval of the solvent control (one-tail, Excel;Microsoft, Redmond, Wash.). AcE was further characterized as increasingadipogenesis better than or equal to the troglitazone positive controlrelative to the solvent response; the student t-test function of Excelwas used for this evaluation.

Results—The positive controls indomethacin and troglitazone inducedlipogenesis to a similar extent in 3T3-L1 cells (FIG. 11). Unexpectedly,the AcE produced an adipogenic response greater than either of thepositive controls indomethacin and troglitazone.

The lipogenic potential demonstrated in 3T3-L1 cells, dimethylsulfoxide-soluble components of an aqueous Acacia sample #4909 extractdemonstrates a potential to increase insulin sensitivity in humans orother animals exhibiting signs or symptoms of insensitivity to insulin.

Example 12 Increased Adiponectin Secretion from Insulin-Resistant 3T3-L1Adipocytes Elicited by a Dimethyl Sulfoxide-Soluble Fraction of anAqueous Extract of Acacia

The Model—The 3T3-L1 murine fibroblast model as described in Example 11was used in these experiments.

Test Materials—Troglitazone was purchased from Cayman Chemical (AnnArbor, Mich.) while methylisobutylxanthine, dexamethasone, and insulinwere obtained from Sigma (St. Louis, Mo.). The test material was a darkbrown powder produced from a 50:50 (v/v) water/alcohol extract of thegum resin of Acacia sample #4909 and was obtained from Bayir Chemicals(No. 68, South Cross Road, Basavanagudi, India). The extract wasstandardized to contain not less than 20% apecatechin. Batch No. ACat/2304 used in this example contained 20.8% apecatechin as determinedby UV analysis. Penicillin, streptomycin, Dulbecco's modified Eagle'smedium (DMEM) was from Mediatech (Herndon, Va.) and 10% FBS-HI (fetalbovine serum-heat inactivated from Mediatech and Hyclone (Logan, Utah).All other standard reagents, unless otherwise indicted, were purchasedfrom Sigma.

Cell culture and Treatment—Culture of the murine fibroblast cell line3T3-L1 to produce Day 6 differentiated adipocytes was performed asdescribed in Example 10. 3T3-L1 cells were seeded at an initial densityof 1×10⁴ cells/cm² in 96-well plates. For two days, the cells wereallowed grow to reach confluence. Following confluence, the cells wereforced to differentiate into adipocytes by the addition ofdifferentiation medium; this medium consisted of (1) 10% FBS/DMEM (highglucose); (2) 0.5 mM methylisobutylxanthine; (3) 0.5 μM dexamethasoneand (4) 10 μg/ml insulin (MDI medium). From Day 3 through Day 5, themedium was changed to post-differentiation medium consisting of 10 μg/mlinsulin in 10% FBS/DMEM.

Assessing the effect of Acacia on insulin-resistant, mature 3T3-L1 cellswas performed using a modification of the procedure described byFasshauer et al. [Fasshauer, et al. Hormonal regulation of adiponectingene expression in 3T3-L1 adipocytes. BBRC 290:1084-1089, (2002)].Briefly, on Day 6, cells were maintained in serum-free media containing0.5% bovine serum albumin (BSA) for three hours and then treated with 1μg insulin/ml plus solvent or insulin plus test material. Troglitazonewas dissolved in dimethyl sulfoxide and added to achieve concentrationsof 5, 2.5, 1.25 and 0.625 μg/ml. The Acacia extract was tested at 50,25, 12.5 and 6.25 μg/ml. Twenty-four hours later, the supernatant mediumwas sampled for adiponectin determination. The complete procedure fordifferentiation and treatment of cells with test materials is outlinedschematically in FIG. 12.

Adiponectin Assay—The adiponectin secreted into the medium wasquantified using the Mouse Adiponectin Quantikine® Immunoassay kit withno modifications (R&D Systems, Minneapolis, Minn.). Information suppliedby the manufacturer indicated that recovery of adiponectin spiked inmouse cell culture media averaged 103% and the minimum detectableadiponectin concentration ranged from 0.001 to 0.007 ng/ml.

Statistical Calculations and Interpretation—All assays were preformed induplicate. For statistical analysis, the effect of Acacia on adiponectinsecretion was computed relative to the solvent control. Differencesbetween the doses were determined using the student's t-test withoutcorrection for multiple comparisons; the nominal five percentprobability of a type I error was selected.

Potency of the test materials was estimated using a modification of themethod of Hofstee [Hofstee, B. H. Non-inverted versus inverted plots inenzyme kinetics. Nature 184:1296-1298, (1959)] for determination of theapparent Michaelis constants and maximum velocities. Substituting{relative adiponectin secretion/[concentration]} for the independentvariable v/[S] and {relative adiponectin secretion} for the dependantvariable {v}, produced a relationship of the form y=mx+b. Maximumadiponectin secretion relative to the solvent control was estimated fromthe y-intercept, while the concentration of test material necessary forhalf maximal adiponectin secretion was computed from the negative valueof the slope.

Results—All concentrations tested for the positive control troglitazoneenhanced adiponectin secretion with maximal stimulation of 2.44-fold at2.5 μg/ml relative to the solvent control in insulin-resistant 3T3-L1cells (FIG. 13). Both the 50 and 25 μg Acacia/ml concentrationsincreased adiponectin secretion relative to the solvent controls 1.76-and 1.70-fold respectively. While neither of these concentrations ofAcacia was equal to the maximal adiponectin secretion observed withtroglitazone, they were comparable to the 1.25 and 0.625 μg/mlconcentrations of troglitazone.

Estimates of maximal adiponectin secretion derived from modified Hofsteeplots indicated a comparable relative increase in adiponectin secretionwith a large difference in concentrations required for half maximalstimulation. Maximum adiponectin secretion estimated from they-intercept for troglitazone and Acacia catechu was, respectively, 2.29-and 1.88-fold relative to the solvent control. However, theconcentration required for stimulation of half maximal adiponectinsecretion in insulin-resistant 3T3-L1 cells was 0.085 μg/ml fortroglitazone and 5.38 μg/ml for Acacia. Computed upon minimumapecatechin content of 20%, this latter figure for Acacia becomesapproximately 1.0 μg/ml.

Based upon its ability to enhance adiponectin secretion ininsulin-resistant 3T3-L1 cells, Acacia, and/or apecatechin, may beexpected to have a positive effect on clinical pathologies in whichplasma adiponectin concentrations are depressed.

Example 13 Increased Adiponectin Secretion from TNFα-Treated 3T3-L1Adipocytes Elicited by a Dimethyl Sulfoxide-Soluble Fraction of anAqueous Extract of Acacia

The Model—The 3T3-L1 murine fibroblast model as described in Example 11was used in these experiments.

Test Materials—Indomethacin, methylisobutylxanthine, dexamethasone, andinsulin were obtained from Sigma (St. Louis, Mo.). The test material wasa dark brown powder produced from a 50:50 (v/v) water/alcohol extract ofthe gum resin of Acacia sample #4909 and was obtained from BayirChemicals (No. 68, South Cross Road, Basavanagudi, India). The extractwas standardized to contain not less than 20% apecatechin. Batch No. ACat/2304 used in this example contained 20.8% apecatechin as determinedby UV analysis. Penicillin, streptomycin, Dulbecco's modified Eagle'smedium (DMEM) was from Mediatech (Herndon, Va.) and 10% FBS (fetalbovine serum) characterized from Mediatech and Hyclone (Logan, Utah).All other standard reagents, unless otherwise indicted, were purchasedfrom Sigma. Cell culture and Treatment—Culture of the murine fibroblastcell line 3T3-L1 to produce Day 3 differentiated adipocytes wasperformed as described in Example 10. 3T3-L1 cells were seeded at aninitial density of 1×10⁴ cells/cm² in 96-well plates. For two days, thecells were allowed grow to reach confluence. Following confluence, thecells were forced to differentiate into adipocytes by the addition ofdifferentiation medium; this medium consisted of (1) 10% FBS/DMEM (highglucose); (2) 0.5 mM methylisobutylxanthine; (3) 0.5 μM dexamethasoneand (4) 10 μg/ml insulin (MDI medium). From Day 3 through Day 5, themedium was changed to post-differentiation medium consisting of 10% FBSin DMEM. On Day 5 the medium was changed to test medium containing 10, 2or 0.5 ng TNFα/ml in 10% FBS/DMEM with or without indomethacin or Acaciaextract. Indomethacin was dissolved in dimethyl sulfoxide and added toachieve concentrations of 5, 2.5, 1.25 and 0.625 μg/ml. The Acaciaextract was tested at 50, 25, 12.5 and 6.25 μg/ml. On Day 6, thesupernatant medium was sampled for adiponectin determination. Thecomplete procedure for differentiation and treatment of cells with testmaterials is outlined schematically in FIG. 14.

Adiponectin Assay—The adiponectin secreted into the medium wasquantified using the Mouse Adiponectin Quantikine® Immunoassay kit withno modifications (R&D Systems, Minneapolis, Minn.). Information suppliedby the manufacturer indicated that recovery of adiponectin spiked inmouse cell culture media averaged 103% and the minimum detectableadiponectin concentration ranged from 0.001 to 0.007 ng/ml.

Statistical Calculations and Interpretation—All assays were preformed induplicate. For statistical analysis, the effect of indomethacin orAcacia catechu on adiponectin secretion was computed relative to thesolvent control. Differences among the doses and test agents weredetermined using the Student's t-test without correction for multiplecomparisons; the nominal five percent probability of a type I error wasselected.

Results—TNFα significantly (p<0.05) depressed adiponectin secretion 65and 29%, respectively, relative to the solvent controls in mature 3T3-L1cells at the 10 and 2 ng/ml concentrations and had no apparent effect onadiponectin secretion at 0.5 ng/ml (FIG. 15). At 10 and 2 ng TNFα/ml,indomethacin enhanced (p<0.05) adiponectin secretion relative to TNFαalone at all doses tested, but failed to restore adiponectin secretionto the level of the solvent control. Acacia treatment in the presence of10 ng TNFα/ml, produced a similar, albeit attenuated, adiponectinincrease relative to that of indomethacin. The differences inadiponectin stimulation between Acacia catechu and indomethacin were 14,20, 32, and 41%, respectively, over the four increasing doses. Since themultiple between doses was the same for indomethacin and Acacia, theseresults suggest that the potency of indomethacin was greater than theactive material(s) in Acacia at restoring adiponectin secretion to3T3-L1 cells in the presence of supraphysiological concentrations ofTNFα.

Treatment of 3T3-L1 cells with 2 ng TNFα and Acacia produced increasesin adiponectin secretion relative to TNFα alone that were significant(p<0.05) at 6.25, 25 and 50 μg/ml. Unlike the 10 ng TNFα/ml treatments,however, the differences between Acacia and indomethacin were smallerand not apparently related to dose, averaging 5.5% over all fourconcentrations tested. As observed with indomethacin, Acacia did notrestore adiponectin secretion to the levels observed in the solventcontrol.

At 0.5 ng TNFα/ml, indomethacin produced a dose-dependant decrease inadiponectin secretion that was significant (p<0.05) at the 2.5 and 5.0μg/ml concentrations. Interestingly, unlike indomethacin, Acacia catechuincreased adiponectin secretion relative to both the TNFα and solventtreated 3T3-L1 adipocytes at 50 μg/ml. Thus, at concentrations of TNFαapproaching physiologic levels, Acacia catechu enhanced adiponectinsecretion relative to both TNFα and the solvent controls and,surprisingly, was superior to indomethacin.

Based upon its ability to enhance adiponectin secretion in TNFα-treated3T3-L1 cells, Acacia catechu, and/or apecatechin, would be expected tohave a positive effect on all clinical pathologies in which TNFα levelsare elevated and plasma adiponectin concentrations are depressed.

Example 14 A Variety of Commercial Acacia Samples Increase Lipogenesisin the 3T3-L1 Adipocyte Model

The Model—The 3T3-L1 murine fibroblast model as described in Example IIwas used in these experiments. All chemicals and procedures used were asdescribed in Example 11 with the exception that only the Oil Red O assaywas performed to assess Acacia catechu-induced, cellular triglyceridecontent. Acacia catechu sample #5669 was obtained from Natural Remedies(364, 2nd Floor, 16th Main, 4th T Block Bangalore, Karnataka 560041India); and samples #4909, #5667, and #5668 were obtained from BayirChemicals (No. 10, Doddanna Industrial Estate, Penya II Stage,Bangalore, 560091 Karnataka, India). Acacia nilotica samples #5639,#5640 and #5659 were purchased from KDN-Vita International, Inc. (121Stryker Lane, Units 4 & 6 Hillsborough, N.J. 08844). Sample #5640 wasdescribed as bark, sample #5667 as a gum resin and sample #5669 asheartwood powder. All other samples unless indicated were described asproprietary methanol extracts of Acacia catechu bark.

Results—All Acacia samples examined produced a positive lipogenicresponse (FIG. 16). The highest lipogenic responses were achieved fromsamples #5669 the heartwood powder (1.27), #5659 a methanol extract(1.31), #5640 a DMSO extract (1.29) and #4909 a methanol extract (1.31).

This example further demonstrates the presence of multiple compounds inAcacia catechu that are capable of positive modification of adipocytephysiology supporting increased insulin actions.

Example 15 A Variety of Commercial Acacia Samples Increase AdiponectinSecretion the TNFα-3T3-L1 Adipocyte Model

The Model—The 3T3-L1 murine fibroblast model as described in Example 11was used in these experiments. Standard chemicals used and treatment ofcells was performed as noted in Examples 11 and 13. Treatment of 3T3-L1adipocytes with TNFα differed from Example 12, however, in that cellswere exposed to 2 or 10 ng TNFα/ml only. On Day 6 culture supernatantmedia were assayed for adiponectin as detailed in Example 12.Formulations of Acacia samples #4909, #5639, #5659, #5667, #5668, #5640,and #5669 were as described in Example 13.

Results—The 2 ng/ml TNFα reduced adiponectin secretion of 3T3-L1adipocytes by 27% from the solvent control, while adiponectin secretionwas maximally elevated 11% from the TNFα solvent control by 1.25 μgindomethacin/mil (Table 12). Only Acacia formulation #5559 failed toincrease adiponectin secretion at any of the four doses tested. Allother formulations of Acacia produced a comparable maximum increase ofadiponectin secretion ranging from 10 to 15%. Differences were observed,however, with regard to the concentrations at which maximum adiponectinsecretion was elicited by the various Acacia formulations. The mostpotent formulation was #5640 with a maximal stimulation of adiponectinstimulation achieved at 12.5 μg/ml, followed by #4909 and #5668 at 25μg/ml and finally #5639, #5667 and #5669 at 50 μg/ml.

TABLE 12 Relative maximum adiponectin secretion from 3T3-L1 adipocyteselicited by various formulations of Acacia in the presence of 2 ngTNFα/ml. Concentration Adiponectin Test Material [μg/ml] Index† 2 ngTNFα/ml ± 95% CI — 1.00 ± 0.05 Solvent control — 1.27* Indomethacin 1.251.11* Acacia catechu #4909 Bark (methanol 25.0 1.15* extract) Acacianilotica #5639 Heartwood (DMSO 50.0 1.14* extract) Acacia nilotica #5659Bark (methanol 25 1.02  extract) Acacia catechu #5667 Bark (methanol50.0 1.10* extract) Acacia catechu #5668 (Gum resin) 25.0 1.15* Acacianilotica #5640 Bark (DMSO 12.5 1.14* extract) Acacia catechu #5669Heartwood powder 50.0 1.14* (DMSO extract) †Adiponectin Index =[Adiponectin]_(Test)/[Adiponectin]_(TNFα control) *Significantlyincreased (p < 0.05) from TNFα solvent response.

The 10 ng/ml TNFα reduced adiponectin secretion of 3T3-L1 adipocytes by54% from the solvent control, while adiponectin secretion was maximallyelevated 67% from the TNFα solvent control by 5.0 μg indomethacin/ml(Table 13). Troglitazone maximally increased adiponectin secretion 51%at the lowest dose tested 0.625 μg/ml. Acacia formulation #5559 producedthe lowest significant increase (p<0.05) of 12% at 25 μg/ml. All otherformulations of Acacia produced a maximum increase of adiponectinsecretion at 50 μg/ml ranging from 17 to 41%. The most potentformulations were #4909 and #5669 with increases in adiponectinsecretion of 41 and 40%, respectively over the TNFα solvent control.

TABLE 13 Relative maximum adiponectin secretion from 3T3-L1 adipocyteselicited by various formulations of Acacia in the presence of 10 ngTNFα/ml. Concentration Adiponectin Test Material [μg/ml] Index† 10 ngTNFα/ml ± 95% CI — 1.00 ± 0.10 Solvent control — 1.54* Indomethacin 5.01.67* Troglitazone 0.625 1.51* Acacia catechu #4909 Bark (methanol 501.41* extract) Acacia nilotica #5639 Heartwood (DMSO 50 1.26* extract)Acacia nilotica #5659 Bark (methanol 25 1.12* extract) Acacia catechu#5667 Bark (methanol 50 1.26* extract) Acacia catechu #5668 (Gum resin)50 1.30* Acacia nilotica #5640 Bark (DMSO 50 1.17* extract) Acaciacatechu #5669 Heartwood powder 50 1.40* (DMSO extract) †AdiponectinIndex = [Adiponectin]_(Test)/[Adiponectin]_(TNFα control) *Significantlyincreased (p < 0.05) from TNFα solvent response.

The observation that different samples or formulations of Acacia elicitsimilar responses in this second model of metabolic syndrome, furtherdemonstrates the presence of multiple compounds in Acacia that arecapable of positive modification of adipocyte physiology supportingincreased insulin actions.

Example 16 Polar and Non-Polar Solvents Extract Compounds from Acaciacatechu Capable of Increasing Adiponectin Secretion in the TNFα/3T3-L1Adipocyte Model

The Model—The 3T3-L1 murine fibroblast model as described in Example 11was used in these experiments. Standard chemicals used are as noted inExamples 11 and 13. 3T3-L1 adipocytes were treated with 10 ng TNFα/ml asdescribed in Example 13. Culture supernatant media were assayed foradiponectin on Day 6 as detailed in Example 13.

Test Materials—Large chips of Acacia catechu sample #5669 heartwood(each chip weighing between 5-10 grams) were subjected to drilling witha ⅝″ metal drill bit using a standard power drill at low speed. The woodshavings were collected into a mortar, and ground into a fine powderwhile frozen under liquid N₂. This powder was then sieved through a 250micron screen to render approximately 10 g of a fine free-flowingpowder.

TABLE 14 Description of Acacia catechu extraction samples for 3T3-L1adiponectin assay. Extraction solvent Weight of extract [mg] PercentExtracted Gastric fluid¹ 16 11 Dimethyl sulfoxide 40 27 Chloroform 0.20.13 Methanol/water pH = 2 95:5 20 13 Water 10 6.7 Ethyl acetate 4 2.7¹Gastric fluid consisted of 2.90 g NaCl, 7.0 ml concentrated, aqueousHCl, 3.2 g pepsin (800-2500 activity units/mg) diluted to 1000 ml withwater. Final pH was 1.2. For this extraction, the gastricfluid-heartwood suspension remained at 40° C. for one hour followed byremoval of the gastric fluid in vacuo. The remaining residue was thendissolved in MeOH, filtered through a 0.45 micron PTFE syringe filterand concentrated in vacuo.

This powder was dispensed into six glass amber vials (150 mg/vial) andextracted at 40° C. for approximately 10 hr with 2 ml of the solventslisted in Table 14. Following this extraction, the heartwood/solventsuspensions were subjected to centrifugation (5800×g, 10 min.). Thesupernatant fractions from centrifugation were filtered through a 0.45micron PTFE syringe filter into separate amber glass vials. Each ofthese samples was concentrated in vacuo. As seen in Table 7, DMSOextracted the most material from the Acacia catechu heartwood andchloroform extracted the least. All extract samples were tested at 50,25, 12.5, and 6.25 μg/ml.

Pioglitazone was obtained as 45 mg pioglitazone tables from a commercialsource as Actos® (Takeda Pharmaceuticals, Lincolnshire, Ill.). Thetablets were ground to a fine powder and tested at 5.0, 2.5, 1.25 and0.625 μg pioglitazone/ml. Indomethacin was also included as anadditional positive control.

Results—Both positive controls pioglitazone and indomethacin increasedadiponectin secretion by adipocytes in the presence of TNFα, 115 and 94%respectively (FIG. 17). Optimal pioglitazone and indomethacinconcentrations were, 1.25 and 2.5 μg/ml respectively. All extracts ofAcacia catechu sample #5669 increased adiponectin secretion relative tothe TNFα treatment. Among the extracts, the DMSO extract was the mostpotent inducer of adiponectin secretion with maximal activity observedat 6.25 μg extract/ml. This result may be due to the ability of DMSO toextract a wide range of materials of varying polarity. An examination ofFIG. 17 indicates that both the water extract (polar compounds) and thechloroform extract (nonpolar compounds) were similar in their ability toincrease adiponectin secretion in the TNFα/3T3-L1 adipocyte model. It isunlikely that these extracts contained similar compounds. This exampleillustrates the ability of solvents with differing polarities to extractcompounds from Acacia catechu heartwood that are capable of increasingadiponectin secretion from adipocytes in the presence of apro-inflammatory stimulus.

Example 17 Acacia catechu Acidic and Basic Fractions are Capable ofIncreasing Adiponectin Secretion in the TNFα/3T3-L1 Adipocyte Model

The Model—The 3T3-L1 murine fibroblast model as described in Example 11was used in these experiments. Standard chemicals used were as noted inExamples 11 and 13. 3T3-L1 adipocytes were treated with 10 ng TNFα/ml asdescribed in Example 13. Culture supernatant media were assayed foradiponectin on Day 6 as detailed in Example 13.

Test Materials—Acacia catechu sample #5669 was extracted according tothe following procedure: Alkaline isopropyl alcohol solution, (1% (v/v)1.5N NaOH in isopropanol) was added to approximately 50 mg of the dryAcacia catechu heartwood powder #5669 in a 50 ml tube. The sample wasthen mixed briefly, sonicated for 30 minutes, and centrifuged for anhour to pellet the remaining solid material. The supernatant liquid wasthen filtered through 0.45 micron filter paper. The pH of the basicisopropanol used was pH 8.0, while the pH of the collected liquid was pH7.0. A portion of the clear, filtered liquid was taken to dryness invacuo and appeared as a white solid. This sample was termed the driedalkaline extract.

The remaining pelleted material was brought up in acidic isopropylalcohol solution, (1% (v/v) 10% HCl in isopropanol) as a red solution.This sample was mixed until the pellet material was sufficientlydispersed in the liquid and then centrifuged for 30 minutes to againpellet the remaining solid. The pale yellow supernatant fluid was passedthrough a 0.45 micron filter paper. The pH of the collected liquid waspH 3.0 and it was found that in raising the pH of the sample to pH 8-9 areddish-brown precipitate was formed (dried precipitate). Theprecipitate was collected and dried, providing a reddish-brown solid.The supernatant liquid was again passed through a 0.45 micron filter toremove any remaining precipitate; this liquid was a deep yellow color.This remaining liquid was taken to dryness resulting in a solid brownsample and termed dried acidic extract. Recoveries for the threefactions are listed in Table 15. All test materials were assayed at 50,25, 12.5 and 6.25 μg/ml, while the pioglitazone positive control wastested at 5.0, 2.5, 1.25 and 0.625 μg/ml.

TABLE 15 Test material recovery from Acacia catechu heartwood powder.Test Material mg collected (% Acacia catechu sample #5669) Driedalkaline extract 0.9 (1.8) Dried precipitate 1.2 (2.4) Dried acidicextract 1.5 (3.0)

Results: TNFα reduced adiponectin secretion by 46% relative to thesolvent control. Maximal restoration of adiponectin secretion bypioglitazone was 1.47 times the TNFα treatment observed at 1.25 μg/ml(Table 16). Of the test materials, only the dried precipitant failed toincrease adiponectin secretion significantly above the TNFα onlycontrol. The acidic extract and heartwood powder (starting material)were similar in their ability to increase adiponectin secretion in thepresence of TNFα, while the alkaline extract increased adiponectinsecretion only at the highest dose of 50 μg/ml.

TABLE 16 Maximum adiponectin secretion elicited over four doses inTNFα/3T3-L1 model. Concentration Test Material [μg/ml] AdiponectinIndex† DMSO Control — 1.86 TNFα ± 95% CI — 1.00 ± 0.11†† Acacia catechusample #5669 6.25 1.14 heartwood powder Dried alkaline extract 50 1.19Dried precipitate 6.25 1.09 Dried acidic extract 6.25 1.16 Pioglitazone1.25 1.47 †Adiponectin Index =[Adiponectin]_(Test)/[Adiponectin]_(TNFα control) ††Values >1.11 aresignificantly different (p < 0.05) from TNFα control.

Example 18 Decreased Interleukin-6 Secretion from TNFα-Treated 3T3-L1Adipoctes by a Dimethyl Sulfoxide-Soluble Fraction of an Aqueous Extractof Acacia

Interleukin-6 (IL-6) is a multifunctional cytokine that plays importantroles in host defense, acute phase reactions, immune responses, nervecell functions, hematopoiesis and metabolic syndrome. It is expressed bya variety of normal and transformed lymphoid and nonlymphoid cells suchas adipocytes. The production of IL-6 is up-regulated by numeroussignals such as mitogenic or antigenic stimulation, lipopolysaccharides,calcium ionophores, cytokines and viruses [Hibi, M., Nakajima, K.,Hirano T. IL-6 cytokine family and signal transduction: a model of thecytokine system. Mol. Med. 74(1):1-12, (January 1996)]. Elevated serumlevels have been observed in a number of pathological conditionsincluding bacterial and viral infection, trauma, autoimmune diseases,malignancies and metabolic syndrome [Arner, P. Insulin resistance intype 2 diabetes—role of the adipokines. Curr Mol. Med.; 5(3):333-9, (May2005)].

The Model—The 3T3-L1 murine fibroblast model as described in Example 11was used in these experiments. Standard chemicals used were as noted inExamples 11 and 13. 3T3-L1 adipocytes were treated with 10 ng TNFα/ml asdescribed in Example 13. Culture supernatant media were assayed foradiponectin on Day 6 as detailed in Example 13.

Test Materials—Indomethacin, methylisobutylxanthine, dexamethasone, andinsulin were obtained from Sigma (St. Louis, Mo.). The test material wasa dark brown powder produced from a 50:50 (v/v) water/alcohol extract ofthe gum resin of Acacia catechu sample #4909 and was obtained from BayirChemicals (No. 68, South Cross Road, Basavanagudi, India). The extractwas standardized to contain not less than 20% apecatechin. Batch No. ACat/2304 used in this example contained 20.8% apecatechin as determinedby UV analysis. Penicillin, streptomycin, Dulbecco's modified Eagle'smedium (DMEM) was from Mediatech (Herndon, Va.) and 10% FBS (fetalbovine serum) characterized from Mediatech and Hyclone (Logan, Utah).All other standard reagents, unless otherwise indicted, were purchasedfrom Sigma.

Interleukin-6 Assay—The IL-6 secreted into the medium was quantifiedusing the Quantikine® Mouse IL-6 Immunoassay kit with no modifications(R&D Systems, Minneapolis, Minn.). Information supplied by themanufacturer indicated that recovery of IL-6 spiked in mouse cellculture media averaged 99% with a 1:2 dilution and the minimumdetectable IL-6 concentration ranged from 1.3 to 1.8 pg/ml. Allsupernatant media samples were assayed undiluted.

Statistical Calculations and Interpretation—All assays were preformed induplicate. For statistical analysis, the effect of Acacia on adiponectinor IL-6 secretion was computed relative to the solvent control.Differences among the doses were determined using the student's t-testwithout correction for multiple comparisons; the nominal five percentprobability of a type I error (one-tail) was selected.

Results—As seen in previous examples, TNFα dramatically reducedadiponectin secretion, while both indomethacin and the Acacia catechuextract increased adiponectin secretion in the presence of TNFα.Although both the indomethacin positive control and Acacia catechuextract demonstrated dose-related increases in adiponectin secretion,neither material restored adiponectin concentrations to those seen inthe dimethyl sulfoxide controls with no TNFα (Table 17). The Acaciacatechu extract demonstrated a potent, dose-related inhibition of IL-6secretion in the presence of TNFα, whereas indomethacin demonstrated noanti-inflammatory effect.

An examination of the ratio of the anti-inflammatory adiponectin to thepro-inflammatory IL-6 resulted in an excellent dose-related increase inrelative anti-inflammatory activity for both indomethacin and the Acaciacatechu extract.

TABLE 17 Decreased IL-6 and increased adiponectin secretion elicited byAcacia catechu sample #4909 in the TNFα/3T3-L1 model. Test ConcentrationAdiponectin IL-6 Material [μg/ml] Index† Index†† Adiponectin/IL-6 DMSO —2.87* 0.46* 6.24* control TNFα control ± 95% — 1.00 ± 0.079 1.00 ± 0.081.00 ± 0.08 CI Indomethacin 5.00 2.69* 1.10* 2.45* 2.50 2.08* 1.04 2.00* 1.25 1.71* 1.01  1.69* 0.625 1.54* 1.37* 1.12* Acacia 50.0 1.51*0.27* 5.55* catechu sample #4909 25.0 1.19* 0.71* 1.68* 12.5 1.13* 0.78*1.45* 6.25 1.15* 0.93  1.23* The Acacia catechu test material orindomethacin was added in concert with 10 ng TNFα/ml to D5 3T3-L1adipocytes. On the following day, supernatant media were sampled foradiponectin and IL-6 determination. All values were indexed to the TNFαcontrol. †Adiponectin Index =[Adiponectin]_(Test)/[Adiponectin]_(TNFα control) ††IL-6 Index =[IL-6_(Test) − IL-6_(Control)]/[IL-6_(TNFα) − IL-6_(Control)]*Significantly different from TNFα control p < 0.05).

Acacia catechu sample #4909 demonstrated a dual anti-inflammatory actionin the TNFα/3T3-L1 adipocyte model. Components of the Acacia catechuextract increased adiponectin secretion while decreasing IL-6 secretion.The overall effect of Acacia catechu was strongly anti-inflammatoryrelative to the TNFα controls. These results support the use of Acaciacatechu for modification of adipocyte physiology to decrease insulinresistance weight gain, obesity, cardiovascular disease and cancer.

Example 19 Effect of a Dimethyl Sulfoxide-Soluble Fraction of an AqueousAcacia Extract on Secretion of Adiponectin, IL-6 and Resistin fromInsulin-Resistant 3T3-L1 Adipocytes

The Model—The 3T3-L1 murine fibroblast model as described in Example 11was used in these experiments. Standard chemicals and statisticalprocedures used were as noted in Examples 11 and 12. I1-6 was assayed asdescribed in Example 18.

Resistin Assay—The amount of resistin secreted into the medium wasquantified using the Quantikine® Mouse Resistin Immunoassay kit with nomodifications (R&D Systems, Minneapolis, Minn.). Information supplied bythe manufacturer indicated that recovery of resistin spiked in mousecell culture media averaged 99% with a 1:2 dilution and the minimumdetectable resistin concentration ranged from 1.3 to 1.8 pg/mil. Allsupernatant media samples were diluted 1:20 with dilution media suppliedby the manufacturer before assay.

Statistical Calculations and Interpretation—All assays were preformed induplicate. For statistical analysis, the effect of Acacia catechu onadiponectin or IL-6 secretion was computed relative to the solventcontrol. Differences among the doses were determined using the Student'st-test without correction for multiple comparisons; the nominal fivepercent probability of a type I error (one-tail) was selected.

Results—Both troglitazone and the Acacia sample #4909 increasedadiponectin secretion in a dose-related manner in the presence of highconcentrations of insulin (Table 18). While Acacia catechu exhibited ananti-inflammatory effect through the reduction of IL-6 at only the 6.25μg/ml, concentration, troglitazone was pro-inflammatory at the 5.00 and1.25 μg/ml concentrations, with no effect observed at the other twoconcentrations. Resistin secretion was increased in a dose-dependentfashion by troglitazone; however, Acacia catechu decreased resistinexpression likewise in a dose-dependent manner.

As seen in Example 18, Acacia catechu sample #4909 again demonstrated adual anti-inflammatory action in the hyperinsulemia/3T3-L1 adipocytemodel. Components of the Acacia catechu extract increased adiponectinsecretion while decreasing IL-6 secretion. Thus, the overall effect ofAcacia catechu was anti-inflammatory relative to the high insulincontrols. The effect of Acacia catechu on resistin secretion in thepresence of high insulin concentrations was contrary to those oftroglitazone: troglitazone increased resistin expression, while Acaciacatechu further decreased resistin expression. These data suggest thatthe complex Acacia catechu extract are not functioning through PPARγreceptors. These results provide further support the use of Acaciacatechu for modification of adipocyte physiology to decrease insulinresistance weight gain, obesity, cardiovascular disease and cancer.

TABLE 18 Effect of Acacia catechu extract on adiponectin, IL-6 andresistin secretion in the insulin resistant 3T3-L1 model. TestConcentration Adiponectin IL-6 Resistin Material [μg/ml] Index† Index††Index††† Insulin — 1.00 ± 0.30* 1.00 ± 0.23 1.00 ± 0.13 controlTroglitazone 5.00 1.47 1.31 1.43 2.50 2.44 1.06 1.22 1.25 1.87 1.46 1.280.625 2.07 1.00 0.89 Acacia 50.0 1.76 1.23 0.50 catechu sample #490925.0 1.70 0.96 0.61 12.5 1.08 0.92 0.86 6.25 1.05 0.64 0.93 The Acaciacatechu test material or indomethacin was added in concert with 166 nMinsulin to D5 3T3-L1 adipocytes. On the following day, supernatant mediawere sampled for adiponectin, IL-6 and resistin determination. Allvalues were indexed to the insulin only control. †Adiponectin Index =[Adiponectin]_(Test)/[Adiponectin]_(Insulin Control) ††IL-6 Index =[IL-6_(Test)]/[IL-6_(Insulin Control)] †††Resistin Index =[Resistin_(Test)]/[Resistin_(Insulin Control)] *Index values representthe mean ± 95% confidence interval computed from residual mean square ofthe analysis of variance. Values greater or less than Insulin control ±95% CI are significantly different with p < 0.05.

Example 20 Increased Lipogenesis in Adipocytes by Phytochemicals Derivedfrom Hops

The Model—The 3T3-L1 murine fibroblast model as described in Example 11was used in these experiments. Standard chemicals and statisticalprocedures used were as noted in Example 11.

Test Materials—The hops phytochemicals used in this testing aredescribed in Table 19 and were acquired from Betatech Hops Products(Washington, D.C., U.S.A.).

TABLE 19 Description of hops test materials. Hops Test MaterialDescription Alpha acid solution 82% alpha acids/2.7% beta acids/2.95%isoalpha acids by volume. Alpha acids include humulone, adhumulone, andcohumulone. Rho isoalpha acids Rho-isohumulone, rho-isoadhumulone, andrho- (RIAA) isocohumulone. Isoalpha acids (IAA) 25.3% isoalpha acids byvolume. Includes cis & trans isohumulone, cis & trans isoadhumulone, andcis & trans isocohumulone. Tetrahydroisoalpha Complex hops - 8.9% THIAAby volume. Includes cis & acids (THIAA) trans tetrahydro-isohumulone,cis & trans tetrahydro- isoadhumulone and cis & transtetrahydro-isocohumulone Hexahydroisoalpha 3.9% THIAA; 4.4% HHIAA byvolume. The HHIAA acids (HHIAA) isomers include hexahydro-isohumulone,hexahydro- isoadhumulone and hexahydro-isocohumulone. Beta acid solution10% beta acids by volume; <2% alpha acids. The beta acids includelupulone, colupulone, adlupulone and prelupulone. Xanthohumol (XN) >80%xanthohumols by weight. Includes xanthohumol, xanthohumol A, xanthohumolB, xanthohumol C, xanthohumol D, xanthohumol E, xanthohumol G,xanthohumol H, desmethylxanthohumol, xanthogalenol,4′-O-methylxanthohumol, 3′-geranylchalconaringenin,3′,5′diprenylchalconaringenin, 5′-prenylxanthohumol, flavokawin,

dihydroxanthohumol, and iso- dehydrocycloxanthohumol hydrate. Spent hopsXanthohumol, xanthohumol A, xanthohumol B, xanthohumol C, xanthohumol D,xanthohumol E, xanthohumol G, xanthohumol H, trans- hydroxyxanthohumol,1″,2″-dihydroxyxanthohumol C, desmethylxanthohumol B,desmethylxanthohumol J, xanthohumol I, desmethylxanthohumol,isoxanthohumol,

 dihydroxanthohumol, diprenylxanthohumol, 5″- hydroxyxanthohumol,5′-prenylxanthohumol, 6,8- diprenylnaringenin, 8-preylnaringenin,6-prenylnaringen, isoxanthohumol, humulinone, cohumulinone, 4-hydroxybenzaldehyde, and sitosterol-3-O-b- glucopyranoside.Hexahydrocolupulone 1% hexahydrocolupulone by volume in KOH

Cell Culture and Treatment—Hops compounds were dissolved in dimethylsulfoxide (DMSO) and added to achieve concentrations of 10, 5, 4 or 2μg/ml at Day 0 of differentiation and maintained throughout thematuration phase (Days 6 or 7). Spent hops was tested at 50 μg/ml.Whenever fresh media were added, fresh test material was also added.DMSO was chosen for its polarity and the fact that it is miscible withthe aqueous cell culture media. As positive controls, indomethacin andtroglitazone were added, respectively, to achieve final concentrationsof 5.0 and 4.4 μg/ml. Differentiated, D6/D7 3T3-L1 cells were stainedwith 0.36% Oil Red 0 or 0.001% BODIPY.

Results—The positive controls indomethacin and troglitazone inducedlipogenesis to a similar extent in 3T3-L1 cells (FIG. 18). Unexpectedly,four of the hops genera produced an adipogenic response in 3T3-L1adipocytes greater than the positive controls indomethacin andtroglitazone. These four genera included isoalpha acids, Rho-isoalphaacids, tetrahydroisoalpha acids, and hexahydroisoalpha acids. Thisfinding is surprising in light of the published report that the bindingof individual isohumulones with PPARγ was approximately one-third toone-fourth that of the potent PPARγ agonist pioglitazone [Yajima, H.,Ikeshima, E., Shiraki, M., Kanaya, T., Fujiwara, D., Odai, H.,Tsuboyama-Kasaoka, N., Ezaki, O., Oikawa, S., and Kondo, K.Isohumulones, bitter acids derived from hops, activate both peroxisomeproliferator-activated receptor alpha and gamma and reduce insulinresistance. J Biol Chem, 279: 33456-33462, (2004)].

The adipogenic responses of xanthohumols, alpha acids and beta acidswere comparable to indomethacin and troglitazone, while spent hops andhexahydrocolupulone failed to elicit a lipogenic response greater thanthe solvent controls.

Based upon their adipogenic potential in 3T3-L1 cells, the positive hopsphytochemical genera in this study, which included isomerized alphaacids, alpha acids and beta acids as well as xanthohumols, may beexpected to increase insulin sensitivity and decrease serumtriglycerides in humans or other animals exhibiting signs or symptoms ofinsensitivity to insulin.

Example 21 Hops Phytochemicals Increase Adiponectin Secretion inInsulin-Resistant 3T3-L1 Adipocytes

The Model—The 3T3-L1 murine fibroblast model as described in Examples 11and 12 were used in this example. Standard chemicals, hops compoundsRIAA, IAA, THIAA, HHIAA, xanthohumols, hexahydrocolupulone, spent hopswere as described, respectively, in Examples 12 and 20.

Cell Culture and Treatment—Cells were cultured as described in Example12 and treated with hops phytochemicals as previously described.Adiponectin assays and statistical interpretations were as described inExample 12. Potency of the test materials was estimated using amodification of the method of Hofstee for determination of the apparentMichaelis constants and maximum velocities. Substituting {relativeadiponectin secretion/[concentration]} for the independent variablev/[S] and {relative adiponectin secretion} for the dependant variable{v}, produced a relationship of the form y=mx+b. Maximum adiponectinsecretion relative to the solvent control was estimated from they-intercept, while the concentration of test material necessary for halfmaximal adiponectin secretion was computed from the negative value ofthe slope.

Results—The positive control troglitazone maximally enhanced adiponectinsecretion 2.44-fold at 2.5 μg/ml over the solvent control ininsulin-resistant 3T3-L1 cells (FIG. 19). All hops phytochemicals testeddemonstrated enhanced adiponectin secretion relative to the solventcontrol, with isoalpha acids producing significantly more adiponectinsecretion than troglitazone (2.97-fold relative to controls). Of thefour doses tested, maximal adiponectin secretion was observed at 5μg/ml, the highest dose, for isoalpha acids, Rho isoalpha acids,hexahydroisoalpha acids and tetrahydroisoalpha acids. For xanthohumols,spent hops and hexahydro colupulone the maximum observed increase inadiponectin secretion was seen at 1.25, 25 and 12.5 μg/ml, respectively.Observed maximal relative adiponectin expression was comparable totroglitazone for xanthohumols, Rho isoalpha acids, and spent hops andless than troglitazone, but greater than control, for hexahydroisoalphaacids, hexahydro colupulone and tetrahydroisoalpha acids.

TABLE 20 Maximum adiponectin secretion and concentration of testmaterial necessary for half maximal adiponectin secretion estimated,respectively, from the y-intercept and slope of Hofstee plots. TestMaterial at Maximum Adiponectin Half Maximal Secretion^([1]) SecretionTest Material [Fold relative to control] [μg/mL] Isoalpha acids 3.170.49 Xanthohumol 2.47 0.037 Rho isoalpha acids 2.38 0.10Troglitazone^([2]) 2.29 0.085 Spent hops 2.21 2.8 Hexahydroisoalphaacids^([2]) 1.89 0.092 Hexahydro colupulone^([2]) 1.83 3.2Tetrahydroisoalpha acids 1.60 0.11 ^([1])Estimated from linearregression analysis of Hofstee plots using all four concentrationstested ^([2])One outlier omitted and three concentrations used fordose-response estimates

As seen in Table 20, estimates of maximal adiponectin secretion derivedfrom modified Hofstee plots (FIG. 20) supported the observations notedabove. y-Intercept estimates of maximum adiponectin secretion segregatedroughly into three groups: (1) isoalpha acids, (2) xanthohumols, Rhoisoalpha acids, troglitazone, and spent hops, and (3) hexahydroisoalphaacids, hexahydro colupulone and tetrahydroisoalpha acids. Theconcentration of test material required for stimulation of half maximaladiponectin secretion in insulin-resistant 3T3-L1 cells, approximately0.1 μg/ml, was similar for troglitazone, Rho isoalpha acids,tetrahydroisoalpha acid and hexahydroisoalpha acids. The concentrationof isoalpha acids at half maximal adiponectin secretion 0.49 μg/ml wasnearly 5-fold greater. Xanthohumols exhibited the lowest dose for halfmaximal adiponectin secretion estimated at 0.037 μg/ml. The highestconcentrations for the estimated half maximal adiponectin secretionvariable were seen for spent hops and hexahydro colupulone,respectively, 2.8 and 3.2 μg/ml.

Based upon their ability to enhance adiponectin secretion ininsulin-resistant 3T3-L1 cells, the positive hops phytochemical generaseen in this study, isoalpha acids, Rho-isoalpha acids,tetrahydroisoalpha acids, hexahydroisoalpha acids, xanthohumols, spenthops and hexahydro colupulone, may be expected to have a positive effecton all clinical pathologies in which plasma adiponectin concentrationsare depressed.

Example 22 Hops Phytochemicals Exhibit Anti-Inflammatory ActivityThrough Enhanced Adiponectin Secretion and Inhibition of Interleukin-6Secretion in Insulin-Resistant 3T3-L1 Adipocytes

The Model—The 3T3-L1 murine fibroblast model as described in Example 11was used in these experiments. Adiponectin and IL-6 were assayed asdescribed, respectively in Examples 12 and 18. Standard chemicals, hopscompounds RIAA, IAA, THIAA, HHIAA, xanthohumols, hexahydrocolupulone,spent hops were as described in Examples 12 and 20.

Statistical Calculations and Interpretation—All assays were preformed induplicate. For statistical analysis, the effect of hops derivatives onadiponectin or IL-6 secretion was computed relative to the solventcontrol. Differences among the doses were determined using analysis ofvariance without correction for multiple comparisons; the nominal fivepercent probability of a type I error was selected.

Results—Troglitazone and all hops derivatives tested increasedadiponectin secretion in the presence of high concentrations of insulin(Table 21). Troglitazone did not decrease IL-6 secretion in this model.In fact, troglitazone, and HHCL exhibited two concentrations in whichIL-6 secretion was increased, while THIAA and spent hops increased IL-6at the highest concentration and had no effect at the otherconcentrations. The effect of other hops derivatives on IL-6 secretionwas generally biphasic. At the highest concentrations tested, RIAA,HHIAA, and XN increased IL-6 secretion; only IAA did not. Significantdecreases in IL-6 secretion were noted for RIAA, IAA, THIAA, and XN.

TABLE 21 Effect of hops compounds on adiponectin and interleukin-6secretion insulin-resistant 3T3-L1 adipocytes. Concentration AdiponectinIL-6 Test Material [μg/ml] Index† Index†† Adiponectin/IL-6 Insulincontrol ± 95% — 1.00 ± 0.30* 1.00 ± 0.23 1.00 ± 0.30 CI Troglitazone5.00 1.47# 1.31# 1.12 2.50 2.44# 1.06 2.30# 1.25 1.87# 1.46# 1.28 0.6252.07# 1.00 2.07# Rho isoalpha acids 5.0 2.42# 1.28# 1.89# (RIAA) 2.52.27# 0.83 2.73# 1.25 2.07# 0.67# 3.09# 0.625 2.09# 0.49# 4.27# Isoalphaacids 5.0 2.97# 0.78 3.81# (IAA) 2.5 2.49# 0.63# 3.95# 1.25 2.44# 0.60#4.07# 0.625 1.73# 0.46# 3.76# Tetrahydroisoalpha 5.0 1.64# 1.58# 1.04acids (THIAA) 2.5 1.42# 0.89 1.60# 1.25 1.55# 0.94 1.65# 0.625 1.35#0.80 1.69# Hexahydroisoalpha 5.0 1.94# 1.49# 1.30# acids (HHIAA) 2.51.53# 0.74# 2.07# 1.25 1.64# 0.67# 2.45# 0.625 1.69# 0.73# 2.32#Xanthohumols 5.0 2.41# 1.23# 1.96# (XN) 2.5 2.11# 0.96 2.20# 1.25 2.50#0.92 2.72# 0.625 2.29# 0.64# 3.58# Hexahydrocolupulone 50.0 1.65# 2.77#0.60# (HHCL) 25.0 1.62# 1.19 1.36# 12.5 1.71# 0.94 1.82# 6.25 1.05 1.001.05 Spent Hops 50.0 1.92# 1.58# 1.22# 25.0 2.17# 0.86 2.52# 12.5 1.84#1.03 1.79# 6.25 1.46# 1.03 1.42# The Acacia catechu test material orindomethacin was added in concert with 166 nM insulin to D5 3T3-L1adipocytes. On the following day, supernatant media were sampled foradiponectin, IL-6 and resistin determination. All values were indexed tothe insulin only control. †Adiponectin Index =[Adiponectin]_(Test)/[Adiponectin]_(Insulin Control) ††IL-6 Index =[IL-6_(Test)]/[IL-6_(Insulin Control)] *Index value is mean ± 95%confidence interval computed from residual mean square of the analysisof variance. For adiponectin or adiponectin/IL-6, values <0.7 or >1.3are significantly different from insulin control and for IL-6, values<0.77 or >1.23 are significantly different from insulin control.#Significantly different from insulin control p < 0.05.

The adiponectin/IL-6 ratio, a metric of overall anti-inflammatoryeffectiveness, was strongly positive (>2.00) for RIAA, IAA HHIA, and XN.THIAA, HHCL and spent hops exhibited positive, albeit lower,adiponectin/IL-6 ratios. For troglitazone the adiponectin/IL-6 ratio wasmixed with a strongly positive response at 2.5 and 0.625 μg/ml and noeffect at 5.0 or 1.25 μg/ml.

The data suggest that the pro-inflammatory effect of hyperinsulinemiacan be attenuated in adipocytes by hops derivatives RIAA, IAA, HHIA,THIAA, XN, HHCL and spent hops. In general, the anti-inflammatoryeffects of hops derivatives in hyperinsulinemia conditionshyperinsulinemia uncomplicated by TNFα were more consistent than thoseof troglitazone.

Example 23 Hops Phytochemicals Increase Adiponectin Secretion inTNFα-Treated 3T3-L1 Adipocytes

The Model—The 3T3-L1 murine fibroblast model as described in Example 11was used in these experiments. Standard chemicals and hops compoundsIAA, RIAA, HHIAA, and THIAA, were as described, respectively, inExamples 13 and 20. Hops derivatives were tested at concentrations of0.625, 1.25, 2.5, and 5.0 μg/ml. Adiponectin was assayed as described inExample 12.

Results—Overnight treatment of day 5 (D5) 3T3-L1 adipocytes with 10 ngTNFα/ml markedly suppressed adiponectin secretion (FIG. 21). The hopsderivatives IAA, RIAA, HHIAA and THIAA all increased adiponectinsecretion relative to the TNFα/solvent control. Linear dose-responsecurves were observed with RIAA and HHIAA resulting in maximal inhibitionat the highest concentration tested 5.0 μg/ml. IAA elicited maximalsecretion of adiponectin at 1.25 μg/ml, while THIAA exhibited acurvilinear response with maximal adiponectin secretion at 5.0 μg/ml.

The ability of hops derivatives IAA, RIAA, HHIAA and THIAA to increaseadipocyte adiponectin secretion in the presence of supraphysiologicalconcentrations of TNFα supports the usefulness of these compounds in theprevention or treatment of inflammatory conditions involving suboptimaladipocyte functioning.

Example 24 Acacia catechu Formulation Synergistic Interaction with HopsDerivatives to Alter Lipogenesis and Adiponectin Secretion in 3T3-L1Adipocytes

The Model—The 3T3-L1 murine fibroblast model as described in Examples 11and 13 was used in these experiments.

Test Chemicals and Treatment—Standard chemicals used were as noted inExamples 11 and 13. 3T3-L1 adipocytes were treated prior todifferentiation as in Example 11 for computing the lipogenic index orwith TNFα as described in Example 12 for assessing the adiponectinindex. Acacia catechu sample H5669 as described in Example 14 was usedwith hops derivatives Rho-isoalpha acids and isoalpha acids aspreviously described. Acacia catechu and the 5:1 and 10:1 combinationsof Acacia:RIAA and Acacia:IAA were tested at 50, 10, 5.0 and 1.0 μg/ml.RIAA and IAA were tested independently at 5.0, 2.5, 1.25 and 0.625μg/ml.

Calculations—Estimates of expected lipogenic response and adiponectinsecretion of the Acacia/hops combinations and determination of synergywere made as previously described.

Results—All combinations tested exhibited lipogenic synergy at one ormore concentrations tested (Table 22). Acacia:RIAA combinations weregenerally more active than the Acacia:IAA combinations with Acacia:RIAA[5:1] demonstrating synergy at all doses and Acacia:RIAA [10:1]synergistic at 10 and 5.0 μg/ml and not antagonistic at anyconcentration tested. The Acacia:IAA [10:1] combination was alsosynergistic at the two mid-doses and showed no antagonism. WhileAcacia:IAA [5:1] was synergistic at the 50 μg/ml concentration, it wasantagonistic at the 5.0 μg/ml dose.

Similarly, all combinations demonstrated synergy with respect toincreasing adiponectin secretion at one or more concentrations tested(Table 23). Acacia:IAA [10:1] exhibited synergy at all doses, whileAcaca:RIAA [5:1] and Acacia:RIAA [10:1] were synergistic at three dosesand antagonistic at one concentration. The Acacia:IAA [5:1] combinationwas synergistic at 1.0 μg/ml and antagonistic at the higher 10 μg/ml.

TABLE 22 Observed and expected lipogenic response elicited by Acaciacatechu and hops derivatives in the insulin-resistant 3T3-l model.Concentration Lipogenic Index† Test Material [μg/ml] Observed ExpectedResult Acacia/RIAA 50 1.05 0.98 Synergy [5:1]¹ 10 0.96 0.89 Synergy 5.00.93 0.90 Synergy 1.0 0.92 0.89 Synergy Acacia/IAA 50 1.06 0.98 Synergy[5:1]² 10 0.93 0.95 No effect 5.0 0.90 0.98 Antagonism 1.0 0.96 0.98 Noeffect Acacia/RIAA 50 0.99 1.03 No effect [10:1]³ 10 1.00 0.90 Synergy5.0 1.00 0.90 Synergy 1.0 0.94 0.89 No effect Acacia/IAA 50 1.37 1.29Synergy [10:1]⁴ 10 1.16 1.15 No effect 5.0 1.08 1.09 No effect 1.0 1.000.99 No effect †Lipogenic Index = [OD]_(Test)/[OD]_(DMSO control).¹Upper 95% confidence limit is 1.03 with least significant difference =0.03. ²Upper 95% confidence limit is 1.03 with least significantdifference = 0.03 ³Upper 95% confidence limit is 1.07 with leastsignificant difference = 0.07. ⁴Upper 95% confidence limit is 1.02 withleast significant difference = 0.02.

TABLE 23 Observed and expected adiponectin secretion elicited by Acaciacatechu and hops derivatives in the TNFα/3T3-l model. ConcentrationAdiponectin Index† Test Material [μg/ml] Observed Expected ResultAcacia/RIAA 50 1.27 1.08 Synergy [5:1]¹ 10 0.99 1.25 Antagonism 5.0 1.020.92 Synergy 1.0 1.19 1.07 Synergy Acacia/IAA 50 1.13 1.16 No effect[5:1]¹ 10 0.92 1.13 Antagonism 5.0 1.04 1.09 No effect 1.0 1.25 1.13Synergy Acacia/RIAA 50 1.29 1.11 Synergy [10:1]² 10 1.07 0.95 Synergy5.0 0.94 1.06 Antagonism 1.0 1.03 0.94 Synergy Acacia/IAA 50 1.28 0.82Synergy [10:1]² 10 1.12 1.07 Synergy 5.0 1.11 0.99 Synergy 1.0 1.30 1.05Synergy †Adiponectin Index =[Adiponectin]_(Test)/[Adiponectin]_(TNFα control) ¹Upper 95% confidencelimit is 1.07 with least significant difference = 0.07. ²Upper 95%confidence limit is 1.03 with least significant difference = 0.03

Combinations of Acacia catechu and the hops derivatives Rho isoalphaacids or isoalpha acids exhibit synergistic combinations and only fewantagonistic combinations with respect to increasing lipid incorporationin adipocytes and increasing adiponectin secretion from adipocytes.

Example 25 Anti-Inflammatory Activity of Hops Derivatives in theLipopolysaccharide/3T3-L1 Adipocyte Model

The Model—The 3T3-L1 murine adipocyte model as described in Examples 11and 13 was used in these experiments.

Test Chemicals and Treatment—Standard chemicals were as noted inExamples 11 and 13, however, 100 ng/ml of bacterial lipopolysaccharide(LPS, Sigma, St. Louis, Mo.) was used in place of TNFα on D5. Hopsderivatives Rho-isoalpha acids and isoalpha acids used were as describedin Example 20. The non-steroidal anti-inflammatory drugs (NSAIDs)aspirin, salicylic acid, and ibuprofen were obtained from Sigma. Thecommercial capsule formulation of celecoxib (Celebrex™, G. D. Searle &Co. Chicago, Ill.) was used and cells were dosed based upon content ofactive ingredient. Hops derivatives, ibuprofen, and celecoxib were dosedat 5.00, 2.50, 1.25 and 0.625 μg/ml. Indomethacin, troglitazone, andpioglitazone were tested at 10, 5.0, 1.0 and 0.50 μg/ml. Concentrationsfor aspirin were 100, 50.0, 25.0 and 12.5 μg/ml, while those forsalicylic acid were 200, 100, 50.0 and 25.0 μg/ml. IL-6 and adiponectinwere assayed and data were analyzed and tabulated as previouslydescribed in Example 18 for IL-6 and Example 13 for adiponectin.

Results—LPS provided a 12-fold stimulation of IL-6 in D5 adipocytes. Alltest agents reduced IL-6 secretion by LPS-stimulated adipocytes tovarying degrees. Maximum inhibition of IL-6 and concentrations for whichthis maximum inhibition were observed are presented in Table 24. Due toa relatively large within treatment variance, the extent of maximuminhibition of IL-6 did not differ among the test materials. The dosesfor which maximum inhibition occurred, however, did differ considerably.The rank order of potency for IL-6 inhibition wasibuprofen>RIAA=IAA>celecoxib>pioglitazone=indomethacin>troglitazone>aspirin>salicylicacid. On a qualitative basis, indomethacin, troglitazone, pioglitazone,ibuprofen and celecoxib inhibited IL-6 secretion at all concentrationstested, while RIAA, IAA, and aspirin did not significantly inhibit IL-6at the lowest concentrations (data not shown).

LPS treatment of D5 3T3-L1 adipocytes decreased adiponectin secretionrelative to the DMSO control (Table 25). Unlike IL-6 inhibition in whichall test compounds inhibited secretion to some extent, aspirin,salicylic acid and celecoxib failed to induce adiponectin secretion inLPS-treated 3T3-L1 adipocytes at any of the does tested. Maximumadiponectin stimulation of 15, 17, 20 and 22% was observed,respectively, for troglitazone, RIAA, IAA and ibuprofen at 0.625 μg/ml.Pioglitazone was next in order of potency with adiponectin stimulationof 12% at 1.25 μg/ml. With a 9% stimulation of adiponectin secretion at2.50 μg/ml, indomethacin was least potent of the active test materials.

In the LPS/3T3-L1 model, hops derivatives RIAA and IAA as well asibuprofen decreased IL-6 secretion and increased adiponectin secretionat concentrations likely to be obtained in vivo. The thiazolidinedionestroglitazone and pioglitazone were less potent as inhibitors of IL-6secretion, requiring higher doses than hops derivatives, but similar tohops derivatives with respect to adiponectin stimulation. No consistentrelationship between anti-inflammatory activity in macrophage models andthe adipocyte model was observed for the NSAIDs indomethacin, aspirin,ibuprofen and celecoxib.

TABLE 24 Maximum inhibition of IL-6 secretion in LPS/3T3-L1 adipocytesby hops derivatives and selected NSAIDs Concentration IL-6 Test Material[μg/ml] Index† % Inhibition DMSO control — 0.09* 91* LPS control ± 95%CI — 1.00 ± 0.30 0 Indomethacin 5.00 0.47* 53* Troglitazone 10.0 0.31*69* Pioglitazone 5.00 0.37* 63* Rho-isoalpha acids 1.25 0.63* 37*Isoalpha acids 1.25 0.61* 39* Aspirin 25.0 0.61* 39* Salicylic acid 50.00.52* 48* Ibuprofen 0.625 0.46* 54* Celecoxib 2.50 0.39* 61* The testmaterials were added in concert with 100 ng LPS/ml to D5 3T3-L1adipocytes. On the following day, supernatant media were sampled forIL-6 determination. All values were indexed to the LPS control as notedbelow. Concentrations presented represent dose providing the maximuminhibition of IL-6 secretion and those values less than 0.70 aresignificantly (p < 0.05) less than the LPS control. †IL-6 Index =[IL-6_(Test) − IL-6_(Control)]/[IL-6_(LPS) − IL-6_(Control)]*Significantly different from LPS control p < 0.05).

TABLE 25 Maximum stimulation of adiponectin secretion in LPS/3T3-L1adipocytes by hops derivatives and selected NSAIDs ConcentrationAdiponectin Test Material [μg/ml] Index† % Stimulation DMSO control —1.24 LPS control ± 95% CI — 1.00 Indomethacin 2.50 1.09*  9 Troglitazone0.625 1.15* 15 Pioglitazone 1.25 1.12* 12 Rho-isoalpha acids 0.625 1.17*17 Isoalpha acids 0.625 1.20* 20 Aspirin 113 1.02 NS Salicylic acid 1730.96 NS Ibuprofen 0.625 1.22* 22 Celecoxib 5.00 1.05 NS †AdiponectinIndex = [Adiponectin]_(Test)/[Adiponectin]_(LPS control) *Values greaterthan 1.07 are significantly different from LPS control p < 0.05). NS =not significantly different from the LPS control.

Example 26 Synergy of Acacia catechu or Hops Derivatives in Combinationwith Curcumin or Xanthohumols in the TNFα/3T3-1 Model

The Model—The 3T3-L1 murine fibroblast model as described in Examples 11and 13 was used in these experiments.

Test Chemicals and Treatment—Standard chemicals used were as noted inExample 11 and 13. 3T3-L1 adipocytes were stimulated with TNFα asdescribed in Example 13 for assessing the adiponectin index. Acaciacatechu sample 45669 as described in Example 14, hops derivativesRho-isoalpha acids and xanthohumol as described in Example 20, andcurcumin as provided by Metagenics (Gig Harbor, Wash.) and were used inthese experiments. Acacia catechu and the 5:1 combinations ofAcacia:curcumin and Acacia:xanthohumol were tested at 50, 10, 5.0 and1.0 μg/ml. RIAA and the 1:1 combinations with curcumin and XN weretested at 10, 5, 1.0 and 0.50 μg/ml.

Calculations—Estimates of expected adiponectin index of the combinationsand determination of synergy were made as described previously.

Results—TNFα reduced adiponectin secretion to about 50 percent ofsolvent only controls. The positive control pioglitazone increasedadiponectin secretion by 80 percent (Table 26). Combinations of Acaciawith curcumin or XN proved to be antagonistic at the higherconcentrations and synergistic at the lower concentrations. Similarly,RIAA and curcumin were antagonistic at the three higher doses, buthighly synergistic at the lowest dose 1.0 μg/ml. The two hops derivativeRIAA and XN did not demonstrate synergy in adiponectin secretion fromTNFα-stimulated 3T3-L1 cells.

In TNFα-treated 3T3-L1 adipocytes, both Acacia and RIAA synergisticallyincreased adiponectin secretion, while only Acacia demonstrated synergywith XN.

TABLE 26 Synergy of Acacia catechu and hops derivatives in combinationswith curcumin or xanthohumols in the TNFα/3T3-l model. Con- centrationAdiponectin Index† Test Material [μg/ml] Observed ExpectedInterpretation DMSO Control — 2.07 — — TNFα ± 95% CI — 1.0 ± 0.049 — —Pioglitazone 1.0 1.80 — — Acacia/Curcumin 50 0.56 0.94 Antagonism [5:1]¹10 1.01 1.07 Antagonism 5.0 1.19 1.02 Synergy 1.0 1.22 1.16 SynergyAcacia/XN [5:1]¹ 50 0.54 0.85 Antagonism 10 0.95 1.06 Antagonism 5.00.97 1.01 Antagonism 1.0 1.26 1.15 Synergy RIAA/Curcumin 5 0.46 0.79Antagonism [1:1]¹ 1 1.03 1.11 Antagonism 5.0 1.12 1.28 Antagonism 1.01.30 1.08 Synergy RIAA/XN [1:1]¹ 50 0.31 0.63 Antagonism 10 0.81 1.06Antagonism 5.0 1.09 1.25 Antagonism 1.0 1.09 1.06 No effect †AdiponectinIndex = [Adiponectin]_(Test)/[Adiponectin]_(TNFα control) ¹95%confidence limits are 0.961 to 1.049 with least significant difference =0.049.

Example 27 In Vitro Synergy of Lipogenesis by Conjugated Linoleic Acidin Combination with Hops Derivative Rho-Isoalpha Acids in theInsulin-Resistant 3T3-L1 Adipocyte Model

The Model—The 3T3-L1 murine fibroblast model as described in Examples 11and 13 was used in these experiments.

Test Chemicals and Treatment—Standard chemicals used were as noted inExample 11. 3T3-L1 adipocytes were treated prior to differentiation asin Example 11 for computing the lipogenic index. Powdered CLA wasobtained from Lipid Nutrition (Channahon, Ill.) and was described as a1:1 mixture of the c9t11 and t10c12 isomers. CLA and the 5:1combinations of CLA:RIAA were tested at 50, 10, 5.0 and 1.0 μg/ml. RIAAwas tested at 10, 1.0 and 0.1 μg/ml for calculation of expectedlipogenic index as described previously.

Results—RIAA synergistically increased triglyceride content incombination with CLA. Synergy was noted at all does (Table 27).

Synergy between CLA and RIAA was observed over a wide range of doses andpotentially could be used to increase the insulin sensitizing potency ofCLA.

TABLE 27 Synergy of lipogenesis by conjugated linoleic acid incombination Rho-isoalpha acids in the insulin-resistant 3T3-L1 adipocytemodel. Lipogenic Index† Concentration Ob- Test Material [μg/ml] servedExpected Interpretation CLA:RIAA[5:1]¹ 50 1.26 1.15 Synergy 10 1.16 1.06Synergy 5.0 1.16 1.10 Synergy 1.0 1.17 1.06 Synergy †Lipogenic Index =[OD]_(Test)/[OD]_(DMSO control). ¹Upper 95% confidence limit is 1.05with least significant difference = 0.05.

Example 28 Hops Phytochemicals Inhibit NF-kB Activation in TNFα-Treated3T3-L1 Adipocytes

The Model—The 3T3-L1 murine fibroblast model as described in Example 11was used in these experiments.

Cell Culture and Treatment—Following differentiation 3T3-L1 adipocyteswere maintained in post-differentiation medium for an additional 40days. Standard chemicals, media and hops compounds RIAA and xanthohumolwere as described in Examples 13 and 20. Hops derivatives and thepositive control pioglitazone were tested at concentrations of 2.5, and5.0 μg/ml. Test materials were added 1 hour prior to and nuclearextracts were prepared three and 24 hours following treatment with TNFα.

ELISA—3T3-L1 adipocytes were maintained in growth media for 40 daysfollowing differentiation. Nuclear NF-kBp65 was determined using theTransAM™ NF-kB kit from Active Motif (Carlsbad, Calif.) was used with nomodifications. Jurkat nuclear extracts provided in the kit were derivedfrom cells cultured in medium supplemented with 50 ng/ml TPA (phorbol,12-myristate, 13 acetate) and 0.5 μM calcium ionophore A23187 for twohours at 37° C. immediately prior to harvesting.

Protein assay—Nuclear protein was quantified using the Active MotifFluorescent Protein Quantification Kit.

Statistical Analysis—Comparisons were performed using a one-tailedStudent's t-test. The probability of a type I error was set at thenominal five percent level.

Results—The TPA-treated Jurkat nuclear extract exhibited the expectedincrease in NF-kBp65 indicating adequate performance of kit reagents(FIG. 22). Treatment of D40 3T3-L1 adipocytes with 10 ng TNFα/ml forthree (FIG. 22A) or 24 hours (FIG. 22B), respectively, increased nuclearNF-kBp65 2.1- and 2.2-fold. As expected, the PPARγ agonist pioglitazonedid not inhibit the amount of nuclear NF-kBp65 at either three or 24hours following TNFα treatment. Nuclear translocation of NF-kBp65 wasinhibited, respectively, 9.4 and 25% at 5.0 and 2.5 μg RIAA/ml at threehours post TNFα. At 24 hours, only the 5.0 RIAA/ml treatment exhibitedsignificant (p<0.05) inhibition of NF-kBp65 nuclear translocation.Xanthohumols inhibited nuclear translocation of NF-kBp65, respectively,15.6 and 6.9% at 5.0 and 2.5 μg/ml at three hours post-TNFα treatmentand 13.4 and 8.0% at 24 hours.

Both RIAA and xanthohumols demonstrated consistent, albeit small,inhibition of nuclear translocation of NF-kBp65 in mature,insulin-resistant adipocytes treated with TNFα. This result differs fromthat described for PPARγ agonists, which have not been shown to inhibitnuclear translocation of NF-kBp65 in 3T3-L1 adipocytes.

Example 29 Acacia catechu Extract and Metformin Synergistically IncreaseTriglyceride Incorporation in Insulin Resistant 3T3-L1 Adipocytes

The Model—The 3T3-L1 murine fibroblast model as described in Example 11was used in these experiments. All chemicals and procedures used were asdescribed in Example 11.

Test Chemicals and Treatment—Metformin was obtained from Sigma (St.Louis, Mo.). Test materials were added in dimethyl sulfoxide at Day 0 ofdifferentiation and every two days throughout the maturation phase (Day6/7). As a positive control, troglitazone was added to achieve a finalconcentration of 4.4 μg/ml. Metformin, Acacia catechu sample #5669 andthe metformin/Acacia combination of 1:1 (w/w) were tested at 50 μg testmaterial/ml. Differentiated 3T3-L1 cells were stained with 0.2% Oil RedO. The resulting stained oil droplets were dissolved with isopropanoland quantified by spectrophotometric analysis at 530 nm. Results wererepresented as a relative triglyceride content of fully differentiatedcells in the solvent controls.

Calculations—An estimate of the expected adipogenic effect of themetformin/Acacia catechu extract was made using the relationship:1/LI=X/LIx+Y/LIy, where LI=the lipogenic index, X and Y were therelative fractions of each component in the test mixture and X+Y=1.Synergy was inferred if the mean of the estimated LI fell outside of the95% confidence interval of the estimate of the corresponding observedfraction. This definition of synergy, involving comparison of theeffects of a combination with that of each of its components, wasdescribed by Berenbaum [Berenbaum, M. C. What is synergy? Pharmacol Rev41(2), 93-141, (1989)].

Results—The Acacia catechu extract was highly lipogenic, increasingtriglyceride content of the 3T3-L1 cells by 32 percent (FIG. 23)yielding a lipogenic index of 1.32. With a lipogenic index of 0.79,metformin alone was not lipogenic. The metformin/Acacia catechu extractcombination demonstrated an observed lipogenic index of 1.35. With anexpected lipogenic index of 98, the metformin/Acacia catechu extractdemonstrated synergy as the observed lipogenic index fell outside of thetwo percent 95% upper confidence limit for the expected value.

Based upon the lipogenic potential demonstrated in 3T3-L1 cells, 1:1combinations of metformin and Acacia catechu extract would be expectedto behave synergistically in clinical use. Such combinations would beuseful to increase the range of positive benefits of metformin therapysuch as decreasing plasma triglycerides or extending the period ofmetformin efficacy.

Example 30 In Vitro Synergies of Lipogenesis by Hops Derivatives andThiazolidinediones in the Insulin-Resistant 3T3-L1 Adipocyte Model

The Model—The 3T3-L1 murine fibroblast model as described in Examples 11and 13 was used in these experiments.

Test Chemicals and Treatment—Standard Chemicals Used Were as Noted inExample 11. 3T3-L1 adipocytes were treated prior to differentiation asin Example 11 for computing the lipogenic index. Troglitazone wasobtained from Cayman Chemicals (Chicago, Ill.). Pioglitazone wasobtained as the commercial, tableted formulation (ACTOSE®, TakedaPharmaceuticals, Lincolnshire, Ill.). The tablets were crushed and thewhole powder was used in the assay. All results were computed based uponactive ingredient content. Hops derivatives Rho-isoalpha acids andisoalpha acids used were as described in Example 20. Troglitazone incombination with RIAA and IAA was tested at 4.0 μg/ml, while the morepotent pioglitazone was tested in 1:1 combinations with RIAA and IAA at2.5 μg/ml. All materials were also tested independently at 4.0 and 2.5μg/ml for calculation of expected lipogenic index as described inExample 34.

Results—When tested at 4.0 and 2.5 μg/ml, respectively, withtroglitazone or piroglitazone, both Rho-isoalpha acids and isoalphaacids increased triglyceride synthesis synergistically with thethiazolidinediones in the insulin-resistant 3T3-L1 adipocyte model(Table 28).

Hops derivatives Rho-isoalpha acids and isoalpha acids couldsynergistically increase the insulin sensitizing effects ofthiazolidinediones resulting in potential clinical benefits ofdose-reduction or increased numbers of patients responding favorably.

TABLE 28 In vitro synergies of hops derivatives and thiazolidinedionesin the insulin-resistant 3T3-L1 adipocyte model. Concentration LipogenicIndex† Test Material 8 μg/ml] Observed Expected InterpretationTroglitazone/ 4.0 1.23 1.06 Synergy RIAA [1:1]¹ Troglitazone/ 4.0 1.141.02 Synergy IAA [1:1]¹ Pioglitazone/ 2.5 1.19 1.00 Synergy RIAA [1:1]²Pioglitazone/ 2.5 1.16 0.95 Synergy IAA [1:1]² †Lipogenic Index =[OD]_(Test)/[OD]_(DMSO control). ¹Upper 95% confidence limit is 1.02with least significant difference = 0.02. ²Upper 95% confidence limit is1.05 with least significant difference = 0.05.

Example 31 In Vitro Synergies of Rho-Isoalpha Acids and Metformin in theTNFα/3T3-L1 Adipocyte Model

The Model—The 3T3-L1 murine fibroblast model as described in Example 11was used in these experiments. Standard chemicals used and treatment ofadipocytes with 10 ng TNFα/ml were as noted, respectively, in Examples11 and 13.

Test Materials and Cell Treatment—Metformin was obtained from Sigma (St.Louis, Mo.) and Rho-isoalpha acids were as described in Example 20.Metformin at 50, 10, 5.0 or 1.0 μg/ml without or with 1 μg RIAA/ml wasadded in concert with 10 ng TNFα/ml to D5 3T3-L1 adipocytes. Culturesupernatant media were assayed for IL-6 on Day 6 as detailed in Example11. An estimate of the expected effect of the metformin:RIAA mixtures onIL-6 inhibition was made as previously described.

Results—TNFα provided a six-fold increase in IL-6 secretion in D5adipocytes. Troglitazone at 1 μg/ml inhibited IL-6 secretion 34 percentrelative to the controls, while 1 μg RIAA inhibited IL-6 secretion 24percent relative to the controls (Table 29). Metformin in combinationwith 1 μg RIAA/ml demonstrated synergy at the 50 μg/ml concentration andstrong synergy at the 1 μg/ml concentration. At 50 μg metformin/mil, 1μg RIAA provided an additional 10 percent inhibition in the mixture;while at 1 μg metformin, 1 μg RIAA increased IL-6 inhibition by 35percent. Antagonism and no effect, respectively, were seen of themetformin:RIAA combinations at the two mid-doses.

Combinations of metformin and Rho-isoalpha acids functionsynergistically at both high and low concentrations to reduce IL-6secretion from TNFα-treated 3T3-L1 adipocytes.

TABLE 29 Synergistic inhibition of IL-6 secretion in TNFα/3T3-L1adipocytes by hops Rho-isoalpha acids and metformin. Concentration %Test Material [μg/ml] IL-6 Index† Inhibition Interpretation DMSO — 0.16— — control TNFα — 1.00 ± 0.07* 0 — control ± 95% CI Troglitazone 1.00.66 34 — RIAA 1.0 0.76 24 — Metformin 50 0.78 22 — Metformin/ 50 0.6832 Synergy 1 μg RIAA Metformin 10 0.78 22 — Metformin/ 10 0.86 14Antagonism 1 μg RIAA Metformin 5.0 0.96 4 — Metformin/ 5.0 0.91 9 Noeffect 1 μg RIAA Metformin 1.0 0.91 9 — Metformin/ 1.0 0.56 44 Synergy 1μg RIAA The test materials were added in concert with 10 ng TNFα/ml toD5 3T3-L1 adipocytes at the stated concentrations. On the following day,supernatant media were sampled for IL-6 determination. All values wereindexed to the TNFα control. †IL-6 Index = [IL-6_(Test) −IL-6_(Control)]/[IL-6_(TNFα) − IL-6_(Control)] *Values less than 0.93are significantly (p < 0.05) less than the TNFα control.

Example 32 Effects of Test Compounds on Cancer Cell Proliferation InVitro

This experiment demonstrates the direct inhibitory effects on cancercell proliferation in vitro for a number of test compounds of theinstant invention.

Methods—The inhibitory effects of test compounds of the presentinvention on cancer cell proliferation were examined in the RL 95-2endometrial cancer cell model (an over expresser of AKT kinase), and inthe HT-29 (constitutively expressing COX-2) and SW480 (constitutivelyexpressing activated AKT kinase) colon cancer cell models. Briefly, thetarget cells were plated into 96 well tissue culture plates and allowedto grow until subconfluent. The cells were then treated for 72 hourswith various amounts of the test compounds as described in Example 4 andrelative cell proliferation determined by the CyQuant (Invitrogen,Carlsbad, Calif.) commercial fluorescence assay.

Results—RL 95-2 cells were treated for 72 hours with 10 μg/ml of MgDHIAA(mgRho), IAA, THIAA, TH-HHIAA (a 1:1 mixture of THIAA & HHIAA), Xn(xanthohumol), LY (LY 249002, a PI3K inhibitor), EtOH (ethanol), alpha(alpha acid mixture), and beta (beta acid mixture). The relativeinhibition on cell proliferation is presented as FIG. 24, showing agreater than 50% inhibition for xanthohumol relative to the DMSO solventcontrol. FIGS. 25 & 26 display the dose response results for variousconcentrations of RIAA or THIAA on HT-29 and SW480 cancer cellsrespectively. Median inhibitory concentrations for RIAA and THIAA were31 and 10 μM for the HT-29 cell line and 38 and 3.2 μM for the SW480cell line.

Example 33 In Vivo Hypoglycemic Action of Acacia nilotica and HopsDerivatives in the KK-A^(y) Mouse Diabetes Model

The Model—Male, nine-week old KK-A^(y)/Ta mice averaging 40±5 grams wereused to assess the potential of the test materials to reduce fastingserum glucose or insulin concentrations. This mouse strain is the resultof hybridization between the KK strain, developed in the 1940s as amodel of diabetes and a strain of A^(y)/a genotype. The observedphenotype is the result of polygenic mutations that have yet to be fullycharacterized but at least four quantitative trait loci have beenidentified. One of these is linked to a missense mutation in the leptinreceptor. Despite this mutation the receptor remains functional althoughit may not be fully efficient. The KK strain develops diabetesassociated with insensitivity to insulin and glucose intolerance but notovert hyperglycemia. Introduction of the A^(y) mutation induces obesityand hyperglycemia. The A^(y) mutation is a 170 kb deletion of the Ralygene that is located 5′ to the agouti locus and places the control foragouti under the Raly promoter. Homozygote animals die beforeimplantation.

Test Materials—Acacia nilotica sample #5659 as described in Example 14and hops derivatives Rho-isoalpha acids, isoalpha acids and xanthohumolsas described in Example 20 were used. The Acacia nilotica, RIAA and IAAwere administered at 100 mg/kg/day, while XN was dosed at 20 mg/kg.Additionally, 5:1 and 10:1 combinations of Acacia nilotica with RIAA,IAA and XN were formulated and dosed at 100 mg/kg/day.

Testing Procedure—Test substances were administered daily by gavage in0.2% Tween-80 to five animals per group. Serum was collected from theretroorbital sinus before the initial dose and ninety minutes after thethird and final dose. Non-fasting serum glucose was determinedenzymatically by the mutarotase/glucose oxidase method and serum insulinwas determined by a mouse specific ELISA (enzyme linked immunosorbentassay).

Data Analysis—To assess whether the test substances decreased eitherserum glucose or insulin relative to the controls, the post-dosingglucose and insulin values were first normalized relative to pre-dosingconcentrations as percent pretreatment for each mouse. The criticalvalue (one-tail, lower 95% confidence interval for the control mice) forpercent pretreatment was computed for both the glucose and insulinvariables. Each percent pretreatment value for the test materials wascompared with the critical value of the control. Those percentpretreatment values for the test materials that were less than thecritical value for the control were considered significantly different(p<0.05) from the control.

Results—During the three-day treatment period, non-fasting, serumglucose rose 2.6% while serum insulin decreased 6.7% in control mice.Rosigltiazone, Acacia nilotica, XN:Acacia [1:5], XN:Acacia [1:10],Acacia:RIAA [5:1], xanthohumols, Acacia:IAA [5:1], isomerized alphaacids and Rho-isoalpha acids all decreased non-fasting serum glucoserelative to the controls with no effect on serum insulin. Acacia:RIAA[10:1] and Acacia:IAA [10:1] had no effect on either serum glucose orinsulin (Table 30).

The rapid hypoglycemic effect of Acacia nilotica sample #5659,xanthohumols, isomerized alpha acids, Rho-isoalpha acids and theirvarious combinations in the KK-Ay mouse model of type 2 diabetessupports their potential for clinical efficacy in the treatment of humandiseases associated with hyperglycemia.

TABLE 30 Effect of Acacia nilotica and hops derivatives on non-fastingserum glucose and insulin in KK-Ay diabetic mice. Dosing† GlucoseInsulin Test Material [mg/kg-day] [% Pretreatment] [% Pretreatment]Control — 102.6 (98.7) 93.3 (85.4) (Critical Value) Rosiglitazone 1.080.3# 88.7 Acacia nilotica 100 89.1# 95.3 sample #5659 XN:Acacia [1:5]100 91.5# 106.5 XN:Acacia [1:10] 100 91.7# 104.4 Acacia:RIAA [5:1] 10092.6# 104.8 Xanthohumols 20 93.8# 106.4 Acacia:IAA [5:1] 100 98.0# 93.2Isomerized alpha 100 98.1# 99.1 acids Rho-isoalpha acids 100 98.3# 100Acacia:RIAA [10:1] 100 101.6 109.3 Acacia:IAA [10:1] 100 104.3 106.4†Dosing was performed once daily for three consecutive days on fiveanimals per group. #Significantly less than control (p < 0.05).

Example 34 In Vivo Synergy of Acacia nilotica and Hops Derivatives inthe Diabetic Db/Db Mouse Model

The Model—Male, C57BLKS/J m⁺/m⁺ Lepr^(db) (db/db) mice were used toassess the potential of the test materials to reduce fasting serumglucose or insulin concentrations. This strain of mice is resistant toleptin by virtue of the absence of a functioning leptin receptor.Elevations of plasma insulin begin at 10 to 14 days and of blood sugarat 4 to 8 weeks. At the time of testing (9 weeks) the animals weremarkedly obese 50±5 g and exhibited evidence of islet hypertrophy.

Test Materials—The positive controls metformin and rosiglitazone weredosed, respectively, at 300 mg/kg-day and 1.0 mg/kg-day for each ofthree consecutive days. Acacia nilotica sample #5659, hops derivativesand their combinations were dosed as described previously.

Testing Piocedure—Test substances were administered daily by gavage in0.2% Tween-80. Serum was collected from the retroorbital sinus beforethe initial dose and ninety minutes after the third and final dose.Non-fasting serum glucose was determined enzymatically by themutarotase/glucose oxidase method and serum insulin was determined by amouse specific ELISA.

Results—The positive controls metformin and rosiglitazone decreased bothserum glucose and insulin concentrations relative to the controls (Table31). Only RIAA and XN demonstrated acceptable results as single testmaterials. RIAA reduced serum insulin, while XN produced a reduction inserum glucose with no effect on insulin. Acacia:RIAA [5:1] was the mosteffective agent tested for reducing serum insulin concentrations,providing a 21 percent reduction in serum insulin levels versus a 17percent reduction in insulin concentrations by the biguanide metforminand a 15 percent decrease by the thiazolidinedione rosiglitazone. Theresponse of this Acacia:RIAA [5:1] combination was greater than theresponses of either individual component thus exhibiting a potential forsynergy. Acacia nilotica alone failed to reduce either serum glucose orinsulin, while RIAA reduced serum insulin to a similar extent asmetformin. Of the remaining test materials, the Acacia:IAA [10:1]combination was also effective in reducing serum insulin concentrations.

The rapid reduction of serum insulin affected by Rho-isoalpha acids andreduction of serum glucose by xanthohumols in the db/db mouse model oftype 2 diabetes supports their potential for clinical efficacy in thetreatment of human diseases associated with insulin insensitivity andhyperglycemia. Further, the 5:1 combination of Rho-isoalpha acids andAcacia catechu appeared synergistic in the db/db murine diabetes model.The positive responses exhibited by Rho-isoalpha acids, xanthohumols andthe Acacia:RIAA [5:1] formulation in two independent animal models ofdiabetes and three in vitro models supports their potential usefulnessin clinical situations requiring a reduction in serum glucose or enhanceinsulin sensitivity.

TABLE 31 Effect of Acacia nilotica and hops derivatives on non-fastingserum glucose and insulin in db/db diabetic mice. Dosing† GlucoseInsulin Test Material [mg/kg-day] [% Pretreatment] [% Pretreatment]Control (Critical — 103.6 (98.4) 94.3 (84.9) Value) Acacia:RIAA [5:1]100 99.6 79.3# Metformin 300 67.6# 83.3# Rho-isoalpha acids 100 102.383.8# Acacia:IAA [10:1] 100 104.3 84.4# Rosiglitazone 1.0 83.0# 84.7#XN:Acacia [1:10] 100 101.5 91.1 Acacia nilotica 100 100.4 91.9sample#5659 Acacia:RIAA [10:1] 100 101.6 93.5 Isomerized alpha 100 100.895.8 acids Xanthohumols 20 97.8# 101.6 XN:Acacia [1:5] 100 104.1 105.6Acacia:IAA [5:1] 100 102.7 109.1 †Dosing was performed once daily forthree consecutive days on five animals per group. #Significantly lessthan respective control (p < 0.05).

Example 35 In Vivo Optimization of Acacia nilotica and Hops DerivativeRatio in the Diabetic db/db Mouse Model

The Model—Male, C57BLKS/J m+/m+Leprdb (db/db) mice were used to assessthe potential of the test materials to reduce fasting serum glucose orinsulin concentrations. This strain of mice is resistant to leptin byvirtue of the absence of a functioning leptin receptor. Elevations ofplasma insulin begin at 10 to 14 days and of blood sugar at 4 to 8weeks. At the time of testing (9 weeks) the animals were markedly obese50±5 g and exhibited evidence of islet hypertrophy.

Test Materials—The positive controls metformin and rosiglitazone weredosed, respectively, at 300 mg/kg-day and 1.0 mg/kg-day for each of fiveconsecutive days. The hops derivative RIAA and Acacia nilotica sample#5659 in ratios of 1:99, 1:5, 1:2, 1:1, 2:1, and 5:1 were dosed at 100mg/kg.

Testing Procedure—Test substances were administered daily by gavage in0.2% Tween-80. Serum was collected from the retroorbital sinus beforethe initial dose and ninety minutes after the fifth and final dose.Non-fasting serum glucose was determined enzymatically by themutarotase/glucose oxidase method and serum insulin was determined by amouse specific ELISA.

Results—The positive controls metformin and rosiglitazone decreased bothserum glucose and insulin concentrations relative to the controls(p<0.05, results not shown). Individually, RIAA and Acacia at 100 mg/kgfor five days reduced serum glucose, respectively, 7.4 and 7.6 percentrelative to controls (p<0.05). Combinations of RIAA and Acacia at 1:99,1:5 or 1:1 appeared antagonistic, while 2:1 and 5:1 ratios ofRIAA:Acacia decreased serum glucose, respectively 11 and 22 percentrelative to controls. This response was greater than either RIAA orAcacia alone and implies a synergic effect between the two components. Asimilar effect was seen with decreases in serum insulin concentrations(FIG. 27).

A 5:1 combination of Rho-isoalpha acids and Acacia was additionallytested in this model against metformin and roziglitazone, twopharmaceuticals currently in use for the treatment of diabetes. Theresults (FIG. 28) indicate that the 5:1 combination of Rho-isoalphaacids and Acacia produced results compatible to the pharmaceuticalagents in reducing serum glucose (panel A) and serum insulin (panel B).

The 2:1 and 5:1 combinations of Rho-isoalpha acids and Acacia appearedsynergistic in the db/db murine diabetes model, supporting theirpotential usefulness in clinical situations requiring a reduction inserum glucose or enhance insulin sensitivity.

Example 36 Effects of Hops Test Compounds in a Collagen InducedRheumatoid Arthritis Murine Model

This example demonstrates the efficacy of two hops compounds, Mg Rho andTHIAA, in reducing inflammation and arthritic symptomology in arheumatoid arthritis model, such inflammation and symptoms being knownto mediated, in part, by a number of protein kinases.

The Model—Female DBA/J mice (10/group) were housed under standardconditions of light and darkness and allow diet ad libitum. The micewere injected intradermally on day 0 with a mixture containing 100 μg oftype II collagen and 100 μg of Mycobacterium tuberculosis in squalene. Abooster injection was repeated on day 21. Mice were examined on days22-27 for arthritic signs with nonresponding mice removed from thestudy. Mice were treated daily by gavage with test compounds for 14 daysbeginning on day 28 and ending on day 42. Test compounds, as used inthis example were RLAA (MgRho) at 10 mg/kg (lo), 50 mg/kg (med), or 250mg/kg (hi); THIAA at 10 mg/kg (lo), 50 mg/kg (med), or 250 mg/kg (hi);celecoxib at 20 mg/kg; and prednisilone at 10 mg/kg.

Arthritic symptomology was assessed (scored 0-4) for each paw using aarthritic index as described below. Under this arthritic index 0=novisible signs; 1=edema and/or erythema: single digit; 2=edema and orerythema: two joints; 3=edema and or erythema: more than two joints; and4=severe arthritis of the entire paw and digits associated withankylosis and deformity.

Results—The effect of RIAA on the arthritic index is presentedgraphically as FIG. 29. Significant reductions (p<0.05,two tail t-test)were observed for prednisolone at 10 mg/kg (days 30-42), celecoxib at 20mg/kg (days 32-42), RIAA at 250 mg/kg (days 34-42) and RIAA at 50 mg/kg(days 38-40), demonstrating antiarthritic efficacy for RIAA at 50 or 250mg/kg. FIG. 30 displays the effects of THIAA on the arthritic index.Here, significant reductions were observed for celecoxib (days 32-42),THIAA at 250 mg/kg (days 34-42) and THIAA at 50 mg/kg (days 34-40), alsodemonstrating the effectiveness of THIAA as an antiarthritic agent.

Example 37 RIAA:Acacia (1:5) Effects on Metabolic Syndrome in Humans

This experiment examined the effects treatment with a RIAA:Acacia (1:5)formulation on a number of clinically relevant markers in volunteerpatients with metabolic syndrome.

Methods and Trial Design—This trial was a randomized,placebo-controlled, double-blind trial conducted at a single study site(the Functional Medicine Research Center, Gig Harbor, Wash.). Inclusioncriteria for the study required subjects (between 18 to 70 years of age)satisfy the following: (i) BMI between 25 and 42.5 kg/m²; (ii) TG/HDL-Cratio≧3.5; (iii) fasting insulin ≧10 mcIU/mL. In addition, subjects hadto meet 3 of the following 5 criteria: (i) waist circumference >35″(women) and >40″ (men); (ii) TG≧150 mg/dL; (iii) HDL<50 mg/dL (women),and <40 mg/dL (men); (iv) blood pressure ≧130/85 or diagnosedhypertension on medication; and (v) fasting glucose ≧100 mg/dL.

Subjects who satisfied the inclusion criteria were randomized to one of4 arms: (i) subjects taking the RIAA/Acacia combination (containing 100mg RIAA and 500 mg Acacia nilotica heartwood extract per tablet) at 1tablet t.i.d.; (ii) subjects taking the RIAA/Acacia combination at 2tablets t.i.d; (iii) placebo, 1 tablet, t.i.d; and (iv) placebo, 2tablets, t.i.d. The total duration of the trial was 12 weeks. Blood wasdrawn from subjects at Day 1, at 8 weeks, and 12 weeks to assess theeffect of supplementation on various parameters of metabolic syndrome.

Results—The initial demographic and biochemical characteristics ofsubjects (pooled placebo group and subjects taking RIAA/Acacia at 3tablets per day) enrolled for the trial are shown in Table 32. Theinitial fasting blood glucose and 2 h post-prandial (2 h pp) glucosevalues were similar between the RAA/Acacia and placebo groups (99.0 vs.96.5 mg/dL and 128.4 vs. 109.2 mg/dL, respectively). In addition, bothglucose values were generally within the laboratory reference range(40-110 mg/dL for fasting blood glucose and 70-150 mg/dL for 2 h ppglucose). This was expected, because alteration in 2 h pp insulinresponse precedes the elevations in glucose and fasting insulin that areseen in later stage metabolic syndrome and frank diabetes.

TABLE 32 Demographic and Baseline Biochemical Characteristics PlaceboRIAA/Acacia (3 tablets/day) N 35 35 Gender Male 11 (31%) 12 (34%) Female24 (69%) 23 (66%) Mean SD Mean SD Age (yrs) 46.0 13.2 47.9 13.4 Weight(lbs) 220.6 35.2 219.5 31.6 BMI (kg/m²) 35.0 4.0 35.4 4.0 Systolic BP(mm) 131.0 15.1 129.7 13.9 Diastolic BP (mm) 83.7 8.5 82.6 7.8 Waist(inches) 42.9 4.9 42.9 4.5 Hip (inches) 47.1 4.0 47.6 3.2 FastingInsulin (mcIU/mL) 13.2 5.2 17.5 12.1 2 h pp Insulin (mcIU/mL) 80.2 52.199.3* 59.2* Fasting Glucose (mg/dL) 96.5 9.0 99.0 10.3 2 h pp Glucose(mg/dL) 109.2 30.5 128.4 36.9 Fasting TG (mg/dL) 231.2 132.2 255.5 122.5*One subject was excluded from the analysis because of abnormal 2 h ppinsulin values; BMI, Basal Metabolic Index; BP, Blood Pressure; TG,Triglyceride; HDL, High-Density Lipoprotein

Fasting blood insulin measurements were similar and generally within thereference range as well, with initial values of 17.5 mcIU/mL for theRIAA/Acacia group, and 13.2 mcIU/mL for the placebo group (referencerange 3-30 mcIU/mL). The 2 h pp insulin levels were elevated past thereference range (99.3 vs. 80.2 mcIU/mL), and showed greater variabilitythan did the fasting insulin or glucose measurements. Although theinitial values were similar, the RIAA/Acacia group showed a greaterdecrease in fasting insulin and 2 h pp insulin, as well as 2 h pp bloodglucose after 8 weeks on the protocol (FIGS. 31 and 32).

The homeostatic model assessment (HOMA) score is a published measure ofinsulin resistance. The change in HOMA score for all subjects is shownin FIG. 33. Due to the variability seen in metabolic syndrome subjects'insulin and glucose values, a subgroup of only those subjects withfasting insulin >15 mcIU/mL was also assessed. The HOMA score for thissubgroup is shown in Table 33, and indicates that a significant decreasewas observed for the RIAA/Acacia group as compared to the placebo group.

TABLE 33 Effect of RIAA/Acacia supplementation (3 tablets/day) on HOMAscores in subjects with initial fasting insulin ≧15 mcIU/mL. HOMA ScoreTreatment N Initial After 8 Weeks Placebo 9 4.39 4.67 RIAA/Acacia 135.84 4.04

The difference between the groups was significant at 8 weeks (p<0.05).HOMA score was calculated from fasting insulin and glucose by publishedmethods [(insulin (mcIU/mL)*glucose (mg/dL))/405].

Elevation in triglycerides (TG) is also an important suggestiveindicator of metabolic syndrome. Table 34 and FIG. 34 indicate thatRIAA/Acacia supplementation resulted in a significant decrease in TGafter 8 weeks as compared with placebo (p<0.05). The TG/HDL-C ratio wasalso shown to decrease substantially for the RIAA/Acacia group (from6.40 to 5.28), while no decrease was noted in the placebo group (from5.81 to 5.92).

TABLE 34 Effect of RIAA/Acacia supplementation (3 tablets/day) on TGlevels and TG/HDL-Cholesterol ratio. Fasting TG (mg/dL) TG/HDL After 8After 8 Supplementation Initial Weeks Change Initial Weeks ChangePlacebo 231.2 229.8 −1.4 5.81 5.92 +0.11 RIAA/Acacia (3 258.6 209.6−49.0 6.40 5.28 −1.12 tablets per day)

Supplementation of metabolic syndrome subjects with a combination tabletcomposed of 100 mg rho-iso-alpha acids and 500 mg Acacia niloticaheartwood extract at 3 tablets per day for a duration of 8 weeks led togreater reduction of 2 h pp insulin levels, as compared to placebo.Further, greater decreases of fasting insulin, fasting and 2 h ppglucose, fasting triglyceride and HOMA scores were observed in subjectstaking RIAA/Acacia supplement (3 tablets per day) versus subjects takingplacebo. These results indicate RIAA/Acacia supplementation might beuseful in maintaining insulin homeostasis in subjects with metabolicsyndrome.

The invention now having been fully described, it will be apparent toone of ordinary skill in the art that many changes and modifications canbe made thereto without departing from the spirit or scope of theappended claims.

1. A method for modulating the activity of a plurality of diseaseassociated protein kinases in a subject in need thereof, wherein saidprotein kinase modulation is beneficial to the health of the subject;said method comprising administering to the subject in need atherapeutically effective amount of a composition comprising a compoundselected from the group consisting of alpha acids, beta acids,prenylflavonoids, chalcones, isoalpha acids, reduced isoalpha acids,spent hops, and a compound or extract derived from acacia.
 2. The methodof claim 1, wherein the subject in need has a condition selected fromthe group consisting of autoimmune disorders, allergic or inflammatorydisorders, metabolic syndrome or diabetes associated disorders, cancer,ocular disorders, and neurological disorders.
 3. The method of claim 2,wherein the disease associated protein kinase is selected from the groupconsisting of ABL, AKT, AURORA, CDK, DBF2/20, EGFR, EPH/ELK/ECK,ERK/MAPKFGFR, GSK3, IKKB, INSR, JAK DOM 1/2, MARK/PRKAA, MEK/STE7,MEKK/STE11, MLK, mTOR, PAK/STE20, PDGFR, PI3K, PKC, POLO, SRC, TEC/ATK,and ZAP/SYK.
 4. The method of claim 1, wherein the alpha acid isselected from the group consisting of humulone, cohumulone, adhumulone,prehumulone, and posthumulone.
 5. The method of claim 1, wherein thebeta acid is selected from the group consisting of lupulone, colupulone,adlupulone, and prelupulone.
 6. The method of claim 1, wherein theisoalpha acid is selected from the group consisting of isohumulone,isoadhumulone, and isocohumulone.
 7. The method of claim 1, wherein thereduced isoalpha acid is selected from the group consisting ofdihydro-isohumulone, dihydro-isocohumulone, dihydro-adhumulone,tetrahydro-isohumulone, tetrahydro-isocohumulone, tetrahydro-adhumulone,hexahydro-isohumulone, hexahydro-isocohumulone, hexahydro-adhumulone,and rho-isoalpha acids.
 8. The method of claim 1, wherein the chalconeis xanthohumol or isoxanthohumol.
 9. The method of claim 1, wherein theprenylflavonoid is 6-prenylnaringenin or 8-prenylnaringenin.
 10. Themethod of claim 1, wherein the compound or extract derived from acaciais derived from Acacia catechu or Acacia nilotica.
 11. The method ofclaim 1, wherein the Acacia catechu or Acacia nilotica compound isselected from the group consisting of gum resin, bark powder, heartwoodpowder, and an Acacia catechu or Acacia nilotica extract.
 12. The methodof claim 1, wherein the Acacia catechu or Acacia nilotica extract isselected from acidic, alkaline, polar solvent, nonpolar solvent, andgastric fluid extracts.
 13. The method of any of claims 1 to 12, whereinthe composition further comprises a pharmaceutically acceptableexcipient selected from the group consisting of coatings, isotonic andabsorption delaying agents, binders, adhesives, lubricants,disintergrants, coloring agents, flavoring agents, sweetening agents,absorbants, detergents, and emulsifying agents.
 14. The method of claim13, wherein the composition further comprises one or more membersselected from the group consisting of antioxidants, vitamins, minerals,proteins, fats, and carbohydrates.
 15. The method of claim 1, whereinthe composition further comprises an antidiabetic drug selected from thegroup consisting of rosiglitazone, troglitazone, pioglitazone, andmetformin.
 16. A method for modulating the activity of a plurality ofmetabolic syndrome associated protein kinases in a subject in needthereof, wherein said protein kinase modulation is beneficial to thehealth of the subject; said method comprising administering to thesubject in need a therapeutically effective amount of a compositioncomprising a 5:1 ratio of rho-isoalpha acids to Acacia niloticaheartwood powder extract.
 17. A composition for modulating the activityof a plurality of disease associated protein kinases in a subject inneed thereof, wherein said protein kinase modulation is beneficial tothe health of the subject; said composition comprising a therapeuticallyeffective amount of a composition comprising a compound selected fromthe group consisting of alpha acids, beta acids, prenylflavonoids,chalcones, isoalpha acids, reduced isoalpha acids, spent hops, and acompound or extract derived from acacia.
 18. The composition of claim17, wherein the subject in need has a condition selected from the groupconsisting of autoimmune disorders, allergic or inflammatory disorders,metabolic syndrome or diabetes associated disorders, cancer, oculardisorders, and neurological disorders.
 19. The composition of claim 17,wherein the disease associated protein kinase is selected from the groupconsisting of ABL, AKT, AURORA, CDK, DBF2/20, EGFR, EPH/ELK/ECK,ERK/MAPKFGFR, GSK3, IKKB, INSR, JAK DOM 1/2, MARK/PRKAA, NMK/STE7,MEKK/STE11, MLK, mTOR, PAK/STE20, PDGFR, PI3K, PKC, POLO, SRC, TEC/ATK,and ZAP/SYK.
 20. The composition of claim 17, wherein the alpha acid isselected from the group consisting of humulone, cohumulone, adhumulone,prehumulone, and posthumulone.
 21. The composition of claim 17, whereinthe beta acid is selected from the group consisting of lupulone,colupulone, adlupulone, and prelupulone.
 22. The composition of claim17, wherein the isoalpha acid is selected from the group consisting ofisohumulone, isoadhumulone, and isocohumulone.
 23. The composition ofclaim 17, wherein the reduced isoalpha acid is selected from the groupconsisting of dihydro-isohumulone, dihydro-isocohumulone,dihydro-adhumulone, tetrahydro-isohumulone, tetrahydro-isocohumulone,tetrahydro-adhumulone, hexahydro-isohumulone, hexahydro-isocohumulone,hexahydro-adhumulone, and rho-isoalpha acids.
 24. The composition ofclaim 17, wherein the chalcone is xanthohumol or isoxanthohumol.
 25. Thecomposition of claim 17, wherein the prenylflavonoid is6-prenylnaringenin or 8-prenylnaringenin.
 26. The composition of claim17, wherein the compound or extract derived from acacia is derived fromAcacia catechu or Acacia nilotica.
 27. The composition of claim 17,wherein the Acacia catechu or Acacia nilotica compound is selected fromthe group consisting of gum resin, bark powder, heartwood powder, and anAcacia catechu or Acacia nilotica extract.
 28. The composition of claim17, wherein the Acacia catechu or Acacia nilotica extract is selectedfrom acidic, alkaline, polar solvent, nonpolar solvent, and gastricfluid extracts.
 29. The composition of any of claims 17 to 28, whereinthe composition further comprises a pharmaceutically acceptableexcipient selected from the group consisting of coatings, isotonic andabsorption delaying agents, binders, adhesives, lubricants,disintergrants, coloring agents, flavoring agents, sweetening agents,absorbants, detergents, and emulsifying agents.
 30. The composition ofclaim 29, wherein the composition further comprises one or more membersselected from the group consisting of antioxidants, vitamins, minerals,proteins, fats, and carbohydrates.
 31. The composition of claim 17,wherein the composition further comprises an antidiabetic drug selectedfrom the group consisting of group rosiglitazone, troglitazone,pioglitazone, and metformin.
 32. A composition for modulating theactivity of a plurality of metabolic syndrome associated protein kinasesin a subject in need thereof, wherein said protein kinase modulation isbeneficial to the health of the subject; said composition comprising atherapeutically effective amount of a composition comprising a 5:1 ratioof rho-isoalpha acids to Acacia nilotica heartwood powder extract.
 33. Acomposition for modulating the activity of a plurality of oculardisorder associated protein kinases in a subject in need thereof,wherein said protein kinase modulation is beneficial to the health ofthe subject; said composition comprising a therapeutically effectiveamount of a composition comprising: a. from about 1 mg to about 1000 mgof vitamin C; b. from about 1 IU to about 1000 IU of vitamin E; c. fromabout 0.1 mg to about 2.5 mg of selenium; d. from about 1 mg to about 50mg of zinc; e. from about 0.1 mg to about 10 mg of copper; f. from about1 mg to about 15 mg of lutein; g. from about 0.05 mg to about 1 mg ofzeaxanthin; and h. from about 1 mg to about 1000 mg of Acacia niloticaheartwood powder extract.
 34. A composition for modulating the activityof a plurality of cancer associated protein kinases in a subject in needthereof, wherein said protein kinase modulation is beneficial to thehealth of the subject; said composition comprising a therapeuticallyeffective amount of at least one member selected from the groupconsisting of: a. from about 0.01 mg to about 10,000 mg of xanthohumol;b. from about 0.01 mg to about 10,000 mg of THIAA; c. from about 0.01 mgto about 10,000 mg of HHIAA; d. from about 0.01 mg to about 10,000 mg ofRIAA; e. from about 0.01 mg to about 10,000 mg of IAA; f. from about0.01 mg to about 10,000 mg of beta acids; and g. from about 0.01 mg toabout 10,000 mg of alpha acids.